Methods and compositions for editing rnas

ABSTRACT

Provided are methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA. Further provided are deaminase-recruiting RNAs used in the RNA editing methods and compositions comprising the same.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to an international application withthe International Application No. PCT/CN2019/082713, filed on Apr. 15,2019, and an international application with the InternationalApplication No. PCT/CN2019/129952, filed on Dec. 30, 2019.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: FD0020PCT-sequencelisting.TXT, date recorded: Apr. 13, 2020, size: 133 KB).

FIELD OF THE INVENTION

The present invention is related to methods and compositions for editingRNAs using an engineered RNA capable of recruiting an adenosinedeaminase to deaminate one or more adenosines in target RNAs.

BACKGROUND OF THE INVENTION

Genome editing is a powerful tool for biomedical research anddevelopment of therapeutics for diseases. So far, the most populargenome editing technology is the Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR)-Cas system, which was developed from theadaptive immune system of bacteria and archaea. CRISPR-Cas can preciselytarget and cleave genome DNA, generating Double-Strand DNA Break (DSB).DSB can be repaired through non-homologous end joining (NHEJ) pathways,and often resulting in an insertion or deletion (Indel), which, in mostcases, inactivates the gene. Alternatively, the homology-directed repair(HDR) pathway can repair the DSB using homologous templates dsDNA orssDNA, and thus, achieve precise genome editing.

Recently, taking advantage of the deaminase proteins, such as AdenosineDeaminase Acting on RNA (ADAR), novel tools were developed for RNAediting. In mammalian cells, there are three types of ADAR proteins,ADAR1 (two isoforms, p110 and p150), ADAR2 and ADAR3 (catalyticallyinactive). The catalytic substrate of ADAR protein is double-strandedRNA.ADAR removes the —NH₂ group from an adenosine (A), converting A toinosine (I), which is recognized as guanosine (G) and paired withcytidine (C) during subsequent cellular transcription and translationprocesses. Researchers fused λN peptide to human ADAR1 or ADAR2deaminase domain to construct the λN-ADARDD system, which could beguided to bind specific RNA targets by a fusion RNA consisting of BoxBstem loop and antisense RNA. This method converts target A to I byintroducing an A-C mismatch at the target A base, resulting in an A to GRNA base editing. Other methods for RNA editing include fusing antisenseRNA to R/G motif (ADAR-recruiting RNA scaffold) to edit target RNA byoverexpressing ADAR1 or ADAR2 protein in mammalian cells, and usingdCas13-ADAR to precisely target and edit RNA. In the application,PCT/EP2017/071912, a method of RNA editing was disclosed which does notrequire exogenous proteins or recruiting domain on nucleic acids. Asynthesized RNA comprising a complementary sequence to the target RNAwas used to induce an A to G base editing. The RNA used in the method isshort (less than 54 nt) and must be specifically modified to increasethe editing efficiency.

SUMMARY OF THE INVENTION

Nucleic acid editing carries enormous potential for biological researchand the development of therapeutics. Most of the current tools for DNAor RNA editing rely on introducing exogenous proteins into livingorganisms, which is subject to potential risks or technical barriers dueto possible aberrant effector activity, delivery limits andimmunogenicity. Some other tools require complicated chemicalmodifications, however still resulting in a low editing efficiency. Insome aspects, the present application provides a programmable approachthat employs a short RNA to leverage a deaminase for targeted RNAediting, in some embodiments, the deaminase is an ADAR (AdenosineDeaminase Acting on RNA) protein, in some embodiments, the ADAR is anendogenous ADAR protein. In some aspects, the present applicationprovides an engineered RNA that is partially complementary to the targettranscript to recruit ADAR1 or ADAR2 to convert adenosine to inosine ata specific site in a target RNA. The methods described herein arecollectively referred to as “LEAPER” (Leveraging Endogenous ADAR forProgrammable Editing on RNA) and the ADAR-recruiting RNAs are referredto interchangeably as “dRNA” or “arRNA”.

In one aspect, the present application provides a method for editing ona target RNA in a host cell, comprising introducing adeaminase-recruiting RNA (dRNA) or a construct encoding thedeaminase-recruiting RNA into the host cell, wherein the dRNA comprisesa complementary RNA sequence that hybridizes to the target RNA, andwherein the dRNA is capable of recruiting an deaminase to deaminate atarget nucleotide, in some embodiments, an adenosine deaminase acting onRNA (ADAR) to deaminate a target adenosine (A) in the target RNA. Incertain embodiments, the host cell is a eukaryotic cell. In someembodiments, the host cell is a mammalian cell. In some embodiments, thehost cell is a human cell. In some embodiments, the host cell is amurine cell. In some embodiments, the host cell is a prokaryotic cell.In some embodiments, the host cell is a primary cell. In someembodiments, the host cell is a T cell.

In certain embodiments, the ADAR is naturally or endogenously present inthe host cell, for example, naturally or endogenously present in theeukaryotic cell. In some embodiments, the ADAR is endogenously expressedby the host cell. In certain embodiments, the ADAR is exogenous to thehost cell. In some embodiments, the ADAR is encoded by a nucleic acid(e.g., DNA or RNA). In some embodiments, the method comprisesintroducing the ADAR or a construct encoding the ADAR into the hostcell. In some embodiments, the method does not comprise introducing anyprotein into the host cell. In certain embodiments, the ADAR is ADAR1and/or ADAR 2. In some embodiments, the ADAR is one or more ADARsselected from the group consisting of hADAR1, hADAR2, murine ADAR1 andmurine ADAR2.

In certain embodiments, the dRNA is not recognized by a Cas(CRISPR-associated protein). In some embodiments, the dRNA does notcomprise crRNA, tracrRNA or gRNA used in a CRISPR/Cas system. In someembodiments, the method does not comprise introducing a Cas or Casfusion protein into the host cell.

In certain embodiments, the deamination of the target A in the targetRNA results in a missense mutation, an early stop codon, aberrantsplicing, or alternative splicing in the target RNA. In someembodiments, the target RNA encodes a protein, and the deamination ofthe target A in the target RNA results in a point mutation, truncation,elongation and/or misfolding of the protein. In some embodiments, thedeamination of the target A in the target RNA results in reversal of amissense mutation, an early stop codon, aberrant splicing, oralternative splicing in the target RNA. In some embodiments, wherein thetarget RNA encodes a truncated, elongated, mutated, or misfoldedprotein, the deamination of the target A in the target RNA results in afunctional, full-length, correctly-folded and/or wild-type protein byreversal of a missense mutation, an early stop codon, aberrant splicing,or alternative splicing in the target RNA. In some embodiments, thetarget RNA is a regulatory RNA, and the deamination of the target Aresults in change in the expression of a downstream molecule regulatedby the target RNA. In certain embodiments, the method is for leveragingan endogenous adenosine deaminase for editing on a target RNA togenerate point mutation and/or misfolding of the protein encoded by thetarget RNA, and/or generating an early stop codon, an aberrant splicesite, and/or an alternative splice site in the target RNA.

In certain embodiments, there is provided a method for editing aplurality of target RNAs in host cells, wherein the method comprisesintroducing a plurality of dRNAs or constructs encoding the a pluralityof dRNAs into the host cells, wherein each of the plurality ofdeaminase-recruiting RNAs comprises a complementary RNA sequence thathybridizes to a corresponding target RNA in the plurality of targetRNAs, and wherein each dRNA is capable of recruiting an adenosinedeaminase acting on RNA (ADAR) to deaminate a target adenosine (A) inthe corresponding target RNA.

In some embodiments, there is provided an edited RNA or a host cellhaving an edited RNA produced by any one of the methods of RNA editingas described above.

In one aspect, the present application provides a method for treating orpreventing a disease or condition in an individual, comprising editing atarget RNA associated with the disease or condition in a cell of theindividual according to any one of the methods for RNA editing asdescribed above. In some embodiments, the method comprises editing thetarget RNA in the cell ex vivo. In some embodiments, the methodcomprises administering the edited cell to the individual. In someembodiments, the method comprises administering to the individual aneffective amount of the dRNA or construct encoding or comprising thedRNA. In some embodiments, the method further comprises introducing tothe cell the ADAR or a construct (e.g., viral vector) encoding the ADAR.In some embodiments, the method further comprises administering to theindividual the ADAR or a construct (e.g., viral vector) encoding theADAR. In some embodiments, the disease or condition is a hereditarygenetic disease. In some embodiments, the disease or condition isassociated with one or more acquired genetic mutations, e.g., drugresistance.

One aspect of the present application provides a dRNA, comprising acomplementary RNA sequence that hybridizes to the target RNA, fordeamination of a target adenosine in a target RNA by recruiting adeaminase, in some embodiments, an Adenosine Deaminase Acting on RNA(ADAR), to deaminate a target adenosine in the target RNA.

In some embodiments according to any one of the methods or dRNAsdescribed herein, the dRNA comprises an RNA sequence comprising acytidine (C), adenosine (A) or uridine (U) directly opposite the targetadenosine to be edited in the target RNA when binding with the targetRNA. The cytidine (C), adenosine (A) and uridine (U) directly oppositethe target adenosine are collectively referred to as “targetingnucleotide”, or separately “targeting C”, “targeting A”, and “targetingU”. In certain embodiments, the RNA sequence further comprises one ormore guanosines each directly opposite a non-target adenosine(s) in thetarget RNA. In certain embodiments, the 5′ nearest neighbor of thetarget A in the target RNA sequence is a nucleotide selected from U, C,A and G with the preference U>C≈A>G and the 3′ nearest neighbor of thetarget A in the target RNA sequence is a nucleotide selected from C, Aand U with the preference G>C>A≈U. In certain embodiments, the target Ais in a three-base motif selected from the group consisting of UAG, UAC,UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAUin the target RNA. In certain embodiments, wherein the three-base motifis UAC; the dRNA comprises an A directly opposite the U in thethree-base motif, a C directly opposite the target A, and a C, G or Udirectly opposite the G in the three-base motif. In certain embodiments,wherein the three-base motif is UAG in the target RNA, the dRNAcomprises ACC, ACG or ACU opposite the UAG of the target RNA.

In some embodiments according to any one of the methods or dRNAsdescribed herein, the deaminase-recruiting RNA comprises more than 40,45, 50, 55, 60, 65, 70, 75 or 80 nucleotides. In certain embodiments,the deaminase-recruiting RNA is 40-260, 45-250, 50-240, 60-230, 65-220,70-210, 70-200, 70-190, 70-180, 70-170, 70-160, 70-150, 70-140, 70-130,70-120, 70-110, 70-100, 70-90, 70-80, 75-200, 80-190, 85-180, 90-170,95-160, 100-150 or 105-140 nucleotides in length. In some embodiments,the dRNA is about 60-200 (such as about any of 60-150, 65-140, 68-130,or 70-120) nucleotides long.

In some embodiments according to any one of the methods or dRNAsdescribed herein, the dRNA described herein can be characterized ascomprising, from 5′ end to 3′ end: a 5′ portion, a cytidine mismatchdirectly opposite to the target A in the target RNA, and a 3′ portion.In some embodiments, the 3′ portion is no shorter than about 7 nt (suchas no shorter than 8 nt, no shorter than 9 nt, and no shorter than 10nt) nucleotides. In some embodiments, the 3′ portion is about 7 nt-25 ntnucleotide long (such as about 8 nt-25 nt, 9 nt-25 nt, 10 nt-25 nt, 11nt-25 nt, 12 nt-25 nt, 13 nt-25 nt, 14 nt-25 nt, 15 nt-25 nt, 16 nt-25nt, 17 nt-25 nt, 18 nt-25 nt, 19 nt-25 nt, 20 nt-25 nt, 21 nt-25 nt, 22nt-25 nt, 23 nt-25 nt, 24 nt-25 nt, and for example, 10 nt-15 nt or 21nt-25 nt nucleotides long). In some embodiments, the 5′ portion is noshorter than about 25 (such as no shorter than about 30, no shorter thanabout 35 nt, no shorter than about 40 nt, and no shorter than about 45nt) nucleotides. In some embodiments, the 5′ portion is about 25 nt-85nt nucleotides long (such as about 25 nt-80 nt, 25 nt-75 nt, 25 nt-70nt, 25 nt-65 nt, 25 nt-60 nt, 30 nt-55 nt, 40 nt-55 nt, or 45 nt-55 ntnucleotides long). In some embodiments, the 5′ portion is about 25 nt-85nt nucleotides long (such as about 25 nt-80 nt, 25 nt-75 nt, 25 nt-70nt, 25 nt-65 nt, 25 nt-60 nt, 30 nt-55 nt, 40 nt-55 nt, or 45 nt-55 ntnucleotides long), and the 3′ portion is about 7 nt-25 nt nucleotidelong (such as about 10 nt-15 nt or 21 nt-25 nt nucleotides long). Insome embodiments, the 5′ portion is longer than the 3′ portion. In someembodiments, the 5′ portion is about 55 nucleotides long, and the 3′portion is about 15 nucleotides long. In some embodiments, the positionof the cytidine mismatch in the dRNA is according to any of the dRNAsdescribed in the examples herein, and the dRNA can be, for example, inthe format of Xnt-c-Ynt, wherein X represents the length of the 5′portion and Y represents the length of the 3′ portion: 55 nt-c-35 nt, 55nt-c-25 nt, 55 nt-c-24 nt, 55 nt-c-23 nt, 55 nt-c-22 nt, 55 nt-c-21 nt,55 nt-c-20 nt, 55 nt-c-19 nt, 55 nt-c-18 nt, 55 nt-c-17 nt, 55 nt-c-16nt, 55 nt-c-15 nt, 55 nt-c-14 nt, 55 nt-c-13 nt, 55 nt-c-12 nt, 55nt-c-11 nt, 55 nt-c-10 nt, 55 nt-c-9 nt, 55 nt-c-8 nt, 55 nt-c-7 nt, 55nt-n-20 nt, 50 nt-n-20 nt, 45 nt-n-20 nt, 55 nt-n-15 nt, 50 nt-n-15 nt,45 nt-c-45 nt, 45 nt-c-55 nt, 54 nt-c-12 nt, 53 nt-c-13 nt, 52 nt-c-14nt, 51 nt-c-15 nt, 50 nt-c-16 nt, 49 nt-c-17 nt, 48 nt-c-18 nt, 47nt-c-19 nt, 46 nt-c-20 nt, 45 nt-c-21 nt, 44 nt-c-22 nt, 43 nt-c-23 nt,54 nt-c-15 nt, 53 nt-c-16 nt, 52 nt-c-17 nt, 51 nt-c-18 nt, 50 nt-c-19nt, 49 nt-c-20 nt, 48 nt-c-21 nt, 47 nt-c-22 nt, 46 nt-c-23 nt, 54nt-c-17 nt, 53 nt-n-18 nt, 52 nt-n-19 nt, 51 nt-n-20 nt, 50 nt-n-21 nt,49 nt-n-22 nt, and 48 nt-c-23.

In certain embodiments, the target RNA is an RNA selected from the groupconsisting of a pre-messenger RNA, a messenger RNA, a ribosomal RNA, atransfer RNA, a long non-coding RNA and a small RNA (e.g., miRNA).

In some embodiments according to any one of the methods or dRNAsdescribed herein, the dRNA is a single-stranded RNA. In someembodiments, the complementary RNA sequence is single-stranded, andwherein the dRNA further comprises one or more double-stranded regions.

In some embodiments, the dRNA comprises one or more modifications, suchas 2′-O-methylation and/or phosphorothioation. In some embodiments, thedRNA is of about 60-200 nucleotides long and comprises one or moremoficiations (such as 2′-O-methylation and/or phosphorothioation). Insome embodiments, the dRNA comprises 2′-O-methylations in the first andlast 3 nucleotides and/or phosphorothiations in the first and last 3internucleotide linkages. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages, and2′-O-methylations in one or more uridines, for example on all uridines.In some embodiments, the dRNA comprises 2′-O-methylations in the firstand last 3 nucleotides, phosphorothiations in the first and last 3internucleotide linkages, 2′-O-methylations in a single or multipleorall uridines, and a modification in the nucleotide opposite to thetarget adenosine, and/or one or two nucleotides most adjacent to thenucleotide opposite to the target adenosine. In certain embodiments, themodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine is a 2′-O-methylation. In certain embodiments, themodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine is a phosphorothiation linkage, such as a3′-phosphorothiation linkage. In certain embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in all uridines, and a 2′-O-methylation in thenucleotide adjacent to the 3′ terminus or 5′ terminus of the nucleotideopposite to the target adenosine. In certain embodiments, the dRNAcomprises 2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in all uridines, and a 3′-phosphorothiation in thenucleotide opposite to the target adenosine and/or its 5′ and/or 3′ mostadjacent nucleotides. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 5 nucleotides andphosphorothiations in the first and last 5 internucleotide linkages.

In certain embodiments according to any one of the methods describedherein, the efficiency of editing on the target RNA is at least about30%, such as at least about any one of 32%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% or higher.

In some embodiments, there is provided a construct (e.g., viral vectoror plasmid) encoding any one of the dRNA described above. In someembodiments, the construct comprises a promoter operably linked to asequence encoding the dRNA. In some embodiments, the construct is a DNAconstruct.

In some embodiments, there is provided a library comprising a pluralityof the dRNAs according to any one of the dRNAs described above or aplurality of the constructs according to any one of the constructsdescribed above.

Also provided are compositions, host cells, kits and articles ofmanufacture comprising any one of the dRNAs described herein, any one ofthe constructs described herein, or any one of the libraries describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H show RNA editing with single dRNA utilizing endogenous ADAR1protein. FIGS. 1A and 1B show schematic representations of RNA editingwith endogenous ADAR1 protein. FIG. 1C shows editing reporter mRNA withdRNA using endogenous ADAR1 protein. FIG. 1D shows statistical analysisof the results in FIG. 1B. FIG. 1E shows ADAR1 knockout and ADAR1(p110),ADAR1(p150) and ADAR2 rescue results. FIG. 1F shows statistical analysisof the results in FIG. 1D. FIG. 1G shows the effect of ADAR1(p110),ADAR1(p150) or ADAR2 overexpression on RNA editing mediated by dRNA in293T-WT cells. FIG. 1H shows that deep sequencing (i.e., Next GenerationSequencing, NGS) results confirmed A to G editing in the targeting site.

FIGS. 2A-2H show optimization of dRNAs. FIG. 2A shows schematicrepresentation of four kinds of base (A, U, C and G) identify oppositeto the targeting adenosine. FIG. 2B shows effects of base identifyopposite to the targeting adenosine on RNA editing efficiency by dRNA.FIG. 2C shows schematic representation of dRNA with one, two or threebases mismatched with UAG targeting site. FIG. 2D shows effects of one,two or three bases mismatched with UAG targeting site on Reporter RNAediting by dRNA. dRNA preferred A-C mismatch on the targeting adenosine.FIG. 2E shows schematic representation of dRNA with variant length. FIG.2F shows the effect of dRNA length on RNA editing efficiency based ondual fluorescence reporter-2. FIG. 2G shows schematic representation ofdifferent A-C mismatch position. FIG. 2H shows effect of A-C mismatchposition on RNA editing efficiency.

FIGS. 3A-3B show editing flexibility for endogenous RNA editing throughexemplary RNA editing method of the present application. FIG. 3A showspercentage quantification of endogenous RNA editing efficiency at all 16different 3-base motifs. FIG. 3B shows heatmap of 5′ and 3′ basepreferences of endogenous RNA editing for 16 different 3 base motifs.

FIGS. 4A-4H show editing the mRNA of endogenous genes with dRNA in 293Tcells. FIG. 4A shows schematic representation of KRAS mRNA target anddRNA with variant length. FIG. 4B shows editing the mRNA of endogenousKRAS gene with dRNA in 293T cells. Empty vector, dRNA-91 nt plasmidswere transfected into 293T-WT cells, respectively. 60 hours later, theRNA was isolated for RT-PCR, and then cDNA was amplified and sequencedon Illumina NextSeq. FIG. 4C shows schematic representation of PPIB mRNAtarget (site1, site2 and site3) and the corresponding dRNA design. FIGS.4D, 4E and 4F show editing the mRNA of endogenous PPIB gene with dRNA in293T cells. FIG. 4G shows schematic representation of β-Actin mRNAtarget and dRNA (71-nt and 131-nt). FIG. 4H shows editing the mRNA ofendogenous β-Actin gene with dRNA in 293T cells.

FIGS. 5A-5G show off-target analysis. FIG. 5A shows schematicrepresentation of the sequence window in which A to I edits wereanalyzed for PPIB mRNA target (PPIB site 1). The black arrow indicatesthe targeted adenosine. FIG. 5B shows deep sequencing quantification ofA to I RNA editing by 151-nt dRNA targeting PPIB mRNA target (PPIB site1). FIG. 5C shows schematic representation of the sequence window inwhich A to I edits were analyzed for KRAS mRNA target. The black arrowindicates the targeted adenosine. FIG. 5D shows deep sequencingquantification of A to I RNA editing by 91-nt and 111-nt dRNA targetingKRAS mRNA target. FIG. 5E shows schematic representation of designedfour kinds of 91-nt or 111-nt dRNA variants containing different A-Gmismatch combinations. The A-G mismatch was designed based on thestatistical results in FIG. 5D and existing knowledge on genic codes fordifferent amino acids. FIG. 5F shows the results of targeted A56 editingby dRNA and different kinds of dRNA variants in FIG. 5E. FIG. 5G showsdeep sequencing quantification of A to I RNA editing by 111-ntdRNA andfour kinds of 111-nt dRNA variants targeting KRAS mRNA target.

FIGS. 6A-6H show RNA editing with single dRNA utilizing endogenous ADAR1protein. FIG. 6A shows schematic representation of RNA editing bydLbuCas13-ADARDD fusion proteins. The catalytically inactive dLbuCas13was fused to the RNA deaminase domains of ADAR1 or ADAR2. FIG. 6B showsschematic representation of dual fluorescence reporter mRNA target andguide RNA design. FIG. 6C shows statistical analysis of the results inFIGS. 6A and 6B. FIG. 6D shows the mRNA level of ADAR1 and ADAR2 in293T-WT cells. FIG. 6E shows genotyping results of ADAR1 gene in293T-ADAR1-KO cell lines by genome PCR. FIG. 6F shows the expressionlevel of ADAR1(p110) and ADAR1(p150) in 293T-WT and 293T-ADAR1-KO celllines via western blotting. FIG. 6G shows the effects of ADAR1(p110),ADAR1(p150) or ADAR2 overexpression on RNA editing mediated by dRNA in293T-WT cells via FACS. FIG. 6H shows Sanger sequencing results showed Ato G editing in the targeted adenosine site.

FIGS. 7A-7C shows optimization of dRNAs. FIG. 7A shows schematicrepresentation of dRNA with variant length and the targeted mRNA editingresults by dRNA with variant length based on dual fluorescencereporter-1. FIG. 7B shows schematic representation of different A-Cmismatch position and the effect of A-C mismatch position on RNA editingefficiency based on dual fluorescence reporter-1. FIG. 7C showsschematic representation of different A-C mismatch position and theeffect of A-C mismatch position on RNA editing efficiency based on dualfluorescence reporter-3.

FIGS. 8A-8B shows editing the mRNA of endogenous genes with dRNA in 293Tcells. FIG. 8A shows editing the mRNA of endogenous β-Actin gene (site2)with dRNA in 293T cells. FIG. 8B shows editing the mRNA of endogenousGAPDH gene with dRNA in 293T cells.

FIG. 9 shows RNA editing by dRNA in different cell lines. FIG. 9A showsthat reporter plasmids and dRNA plasmids were co-transfected intodifferent cell lines, and the results showed that dRNA could functionwell in multiple cell lines, indicating the universality of dRNAapplication.

FIGS. 10A-10D show exploration of an efficient exemplary RNA editingplatform. FIG. 10A, Schematic of dLbuCas13a-ADAR1_(DD) (E1008Q) fusionprotein and the corresponding crRNA. The catalytic inactive LbuCas13awas fused to the deaminase domain of ADAR1 (hyperactive E1008Q variant)using 3×GGGGS linker. The crRNA (crRNA^(Cas13a)) consisted of Lbu-crRNAscaffold and a spacer, which was complementary to the targeting RNA withan A-C mismatch as indicated. FIG. 10B, Schematic of dual fluorescentreporter system and the Lbu-crRNA with various lengths of spacers asindicated. FIG. 10C, Quantification of the EGFP positive (EGFP⁺) cells.HEK293T cells stably expressing the Repoter-1 were transfected withindicated lengths of crRNA^(Cas13a), with or without co-expression ofthe dLbuCas13a-ADAR1_(DD) (E1008Q), followed by FACS analysis. Data arepresented as the mean±s.e.m. (n=3). FIG. 10D, Representative FACS resultfrom the experiment performed with the control (Ctrl crRNA₇₀) or thetargeting spacer (crRNA₇₀).

FIGS. 11A-11G show exemplary methods of leveraging endogenous ADAR1protein for targeted RNA editing. FIG. 11A, Schematic of the Reporter-1and the 70-nt arRNA. FIG. 11B, Representative FACS analysis ofarRNA-induced EGFP expression in wild-type (HEK293T, upper) or ADAR1knockout (HEK293T ADAR1^(−/−), lower) cells stably expressing theRepoter-1. FIG. 11C, Western blot analysis showing expression levels ofADAR1 proteins in wild-type and HEK293T ADAR1^(−/−) cells, as well asthose in HEK293T ADAR1^(−/−) cells transfected with ADAR1 isoforms (p110and p150). FIG. 11D, Western blot analysis showing expression levels ofADAR2 proteins in wild-type and HEK293T ADAR1^(−/−) cells, as well asthose in HEK293T ADAR1^(−/−) cells transfected with ADAR2. FIG. 11E,Quantification of the EGFPpositive (EGFP⁺) cells. Reporter-1 andindicated ADAR-expressing constructs were co-transfected into HEK293TADAR1^(−/−) cells, along with the Ctrl RNA₇₀ or with the targetingarRNA₇₀, followed by FACS analysis. EGFP⁺ percentages were normalized bytransfection efficiency, which was determined by mCherry⁺. Data are meanvalues f s.e.m. (n=4). FIG. 11F, The Electropherograms showing Sangersequencing results in the Ctrl RNA₇₀ (upper) or the arRNA₇₀(lower)-targeted region. FIG. 11G, Quantification of the A to Iconversion rate at the targeted site by deep sequencing.

FIGS. 12A-12B show mRNA expression level of ADAR1/ADAR2 andarRNA-mediated RNA editing. FIG. 12A, Quantitative PCR showing the mRNAlevels of ADAR1 and ADAR2 in HEK293T cells. Data are presented as themean±s.e.m. (n=3). FIG. 12B, Representative FACS results from FIG. 1 e.

FIG. 13 shows quantitative PCR results demonstrating the effects of anexemplary LEAPER method on the expression levels of targeted Reporter-1transcripts by 111-nt arRNA or control RNA in HEK293T cells. Data arepresented as the mean±s.e.m. (n=3); unpaired two-sided Student's t-test,ns, not significant.

FIGS. 14A-14D show targeted RNA editing with an exemplary LEAPER methodin multiple cell lines. FIG. 14A, Western-blot results showing theexpression levels of ADAR1, ADAR2 and ADAR3 in indicated human celllines. β-tubulin was used as a loading control. Data shown is therepresentative of three independent experiments. ADAR1^(−/−)/ADAR2represents ADAR1-knockout HEK293T cells overexpressing ADAR2. FIG. 14B,Relative ADAR protein expression levels normalized by β-tubulinexpression. FIG. 14C, Indicated human cells were transfected withReporter-1, along with the 71-nt control arRNA (Ctrl RNA₇₁) or with the71-nt targeting arRNA (arRNA₇₁) followed by FACS analysis. FIG. 14D,Indicated mouse cell lines were analyzed as described in FIG. 14C. EGFP⁺percentages were normalized by transfection efficiency, which wasdetermined by mCherry⁺. Error bars in FIGS. 14 b, 14C, and 14D allindicate the mean±s.e.m. (n=3); unpaired two-sided Student's t-test,*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant.

FIGS. 15A-15C show schematics of Reporter-1 (FIG. 15A), -2 (FIG. 15B),and -3 (FIG. 15C), as well as their corresponding arRNAs.

FIGS. 16A-16G show characterization and optimization of exemplary LEAPERmethods. FIG. 16A, Top, schematic of the design of arRNAs with changedtriplet (5′-CNA, N denotes A, U, C or G) opposite to the target UAG.Bottom, EGFP⁺ percent showing the effects of variable bases opposite tothe targeted adenosine on RNA editing efficiency. FIG. 16B, Top, thedesign of arRNAs with changed neighboring bases flanking the cytidine inthe A-C mismatch (5′-N¹CN²). Bottom, the effects of 16 differentcombinations of N¹CN² on RNA editing efficiency. FIG. 16C, Summary ofthe preference of 5′ and 3′ nearest neighboring sites of the cytidine inthe A-C mismatch. FIG. 16D, Top, the design of arRNAs with variablelength. Bottom, the effect of arRNA length on RNA editing efficiencybased on Reporter-1 and Reporter-2. FIG. 16E, Top, the design of arRNAswith variable A-C mismatch position. Bottom, the effect of A-C mismatchposition on RNA editing efficiency based on Reporter 1 and Reporter-2.FIG. 16F, Top, the design of the triplet motifs in the reporter-3 withvariable nearest neighboring bases surrounding the targeting adenosine(5′-N¹AN²) and the opposite motif (5′-N²CN¹) on the 111-nt arRNA(arRNA₁₁₁). Bottom, deep sequencing results showing the editing rate ontargeted adenosine in the 5′-N¹AN² motif. FIG. 16G, Summary of the 5′and 3′ base preferences of LEAPER-mediated editing at the Reporter-3.Error bars in FIGS. 16A, 16B, 16D, 16E and 16F all indicate meanvalues±s.e.m. (n=3).

FIGS. 17A-17I show editing of endogenous transcripts with exemplaryLEAPER methods. FIG. 17A, Schematic of the targeting endogenoustranscripts of four disease-related genes (PPIB, KRAS, SMAD4 and FANCC)and the corresponding arRNAs. FIG. 17B, Deep sequencing results showingthe editing rate on targeted adenosine of the PPIB, KRAS, SMAD4 andFANCC transcripts by introducing indicated lengths of arRNAs. FIG. 17C,Deep sequencing results showing the editing rate on non-UAN sites ofendogenous PPIB, FANCC and IDUA transcripts. FIG. 17D, Multiplex editingrate by two 111-nt arRNAs. Indicated arRNAs were transfected alone orwere co-transfected into the HEK293T cells. The targeted editing at thetwo sites was measured from co-transfected cells. FIG. 17E, Schematic ofthe PPIB transcript sequence covered by the 151-nt arRNA. The blackarrow indicates the targeted adenosine. All adenosines were marked inred. FIG. 17F, Heatmap of editing rate on adenosines covered byindicated lengths of arRNAs targeting the PPIB gene (marked in boldframe in blue). For the 111-nt arRNA or arRNA₁₅₁-PPIB covered region,the editing rates of A22, A30, A33, and A34 were determined by RNA-seqbecause of the lack of effective PCR primers for amplifying this region.Otherwise the editing rate was determined by targeted deep-sequencinganalysis. FIG. 17G, Top, the design of the triplet motifs in thereporter-3 with variable nearest neighboring bases surrounding thetargeting adenosine (5′-N¹AN²) and the opposite motif (5′-N^(2′)GN^(1′))in the 111-nt arRNA (arRNA₁₁₁) Bottom, deep sequencing results showingthe editing rate. FIG. 17H, Top, the design of arRNAs with twoconsecutive mismatches in the 5′-N′GN² motif opposite to the 5′-UAG orthe 5′-AAG motifs. Deep sequencing results showing the editing rate byan arRNA₁₁₁ with two consecutive mismatches in the 5′-N¹GN² motifopposite to the 5′-UAG motif (bottom left) or the 5′-AAG motif (bottomright). FIG. 17I, Heatmap of the editing rate on adenosines covered byengineered arRNA₁₁₁ variants targeting the KRAS gene. Data in FIGS. 17B,17C, 17D, 17G and 17H are presented as the mean±s.e.m. (n=3); unpairedtwo-sided Student's t-test, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001;NS, not significant. Data in (f and i) are presented as the mean (n=3).

FIGS. 18A-18B show effects of exemplary LEAPER methods on the expressionlevels of targeted transcripts and protein products. FIG. 18A,Quantitative PCR showing the expression levels of targeted transcriptsfrom PPIB, KRAS, SMAD4 and FANCC by the corresponding 151-nt arRNA orControl RNA in HEK293T cells. Data are presented as the mean±s.e.m.(n=3); unpaired two-sided Student's t-test, *P<0.05; **P<0.01;***P<0.001; ****P<0.0001; ns, not significant. FIG. 18B, Western blotresults showing the effects on protein products of targeted KRAS gene by151-nt arRNA in HEK293T cells. β-tubulin was used as a loading control.

FIGS. 19A-19F show editing of endogenous transcripts with exemplaryLEAPER methods. FIG. 19A, Schematic of the KARS transcript sequencecovered by the 151-nt arRNA. The arrow indicates the targetingadenosine. All adenosines were marked in red. FIG. 19B, Heatmap ofediting rate on adenosines covered by indicated arRNAs in the KARStranscript (marked in the bold frame in blue). FIG. 19C, Schematic ofthe SMAD4 transcript covered by the 151-nt arRNA. FIG. 19D, Heatmap ofediting rate on adenosines covered by indicated arRNAs in the SMAD4transcript. FIG. 19E, Schematic of the FANCC transcript covered by the151-nt arRNA. FIG. 19F, Heatmap of editing rate on adenosines covered byindicated arRNAs in the FANCC transcript. For each arRNA, the region ofduplex RNA is highlighted with bold frame in blue. Data (FIGS. 19B, 19D,and 19F) are presented as the mean (n=3).

FIGS. 20A-20D show transcriptome-wide specificity of RNA editing byLEAPER. FIGS. 20A and 20B, Transcriptome-wide off-targeting analysis ofCtrl RNA₁₅₁ and arRNA₁₅₁-PPIB. The on-targeting site (PPIB) ishighlighted in red. The potential off-target sites identified in bothCtrl RNA and PPIB-targeting RNA groups are labeled in blue. FIG. 20C,The predicted annealing affinity between off-target sites and thecorresponding Ctrl RNA₁₅₁ or arRNA₁₅₁-PPIB. The minimum free energy (AG)of double-stranded RNA formed by off-target sites (150-nt upstream anddownstream of the editing sites) and the corresponding Ctrl RNA₁₅₁ orarRNA₁₅₁-PPIB was predicted with RNAhybrid, an online website tool. FIG.20D, Top, schematic of the highly complementary region betweenarRNA₁₅₁-PPIB and the indicated potential off-target sites, which werepredicted by searching homologous sequences through NCBI-BLAST. Bottom,Deep sequencing showing the editing rate on the on-target site and allpredicted off-target sites of arRNA₁₅₁-PPIB. Data are presented as themean±s.e.m. (n=3).

FIGS. 21A-21B show evaluation of potential off-targets. FIG. 21A,Schematic of the highly complementary region of arRNA₁₁₁-FANCC and theindicated potential off-target sequence, which were predicted bysearching homologous sequences through NCBI-BLAST. FIG. 21B, Deepsequencing showing the editing rate on the on-target site and allpredicted off-target sites of arRNA₁₁₁-FANCC. All data are presented asthe mean±s.e.m. (n=3).

FIGS. 22A-22F show safety evaluation of applying exemplary LEAPERmethods in mammalian cells. FIGS. 22A and 22B, Transcriptome-wideanalysis of the effects of Ctrl RNA₁₅₁ (a) arRNA₁₅₁-PPIB (b) on nativeediting sites by transcriptome-wide RNA-sequencing. Pearson'scorrelation coefficient analysis was used to assess the differential RNAediting rate on native editing sites. FIGS. 22C and 22D, Differentialgene expression analysis of the effects of Ctrl RNA₁₅₁ (c) arRNA₁₅₁-PPIB(d) with RNA-seq data at the transcriptome level. Pearson's correlationcoefficient analysis was used to assess the differential geneexpression. FIGS. 22E and 22F, Effect of arRNA transfection on innateimmune response. The indicated arRNAs or the poly(LC) were transfectedinto HEK293T cells. Total RNA was then analyzed using quantitative PCRto determine expression levels of IFN-β(e) and IL-6 (f). Data (e and f)are presented as the mean±s.e.m. (n=3).

FIGS. 23A-23D show recovery of transcriptional regulatory activity ofmutant TP53W53X by LEAPER. FIG. 23A, Top, Schematic of the TP53transcript sequence covered by the 111-nt arRNA containing c.158G>Aclinical-relevant non-sense mutation (Trp53Ter). The black arrowindicates the targeted adenosine. All adenosines were marked in red.Bottom, the design of two optimized arRNAs targeting TP53^(W53X)transcripts with A-G mismatch on A^(46th) for arRNA₁₁₁-AG1, and onA^(16th), A^(46th), A^(91th) and A^(94th) together for arRNA₁₁₁-AG4 tominimize the potential off-targets on “editing-prone” motifs. FIG. 23B,Deep sequencing results showing the targeted editing on TP53^(W53X)transcripts by arRNA₁₁₁, arRNA₁₁₁-AG1 and arRNA₁₁₁-AG4. FIG. 23C,Western blot showing the recovered production of full-length p53 proteinfrom the TP53^(W53X) transcripts in the HEK293T TP53^(−/−) cells. FIG.23D, Detection of the transcriptional regulatory activity of restoredp53 protein using a p53-Firefly-luciferase reporter system, normalizedby co-transfected Renilla-luciferase vector. Data (b, c and d) arepresented as the mean±s.e.m. (n=3); unpaired two-sided Student's t-test,*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant.

FIG. 24 show editing of mutant TP53W53X transcripts by an exemplaryLEAPER method. Top, schematic of the TP53 transcript sequence covered bythe 111-nt arRNAs. The arrow indicates the targeted adenosine. Alladenosines were marked in red. Bottom, a heatmap of editing rate onadenosines covered by indicated arRNAs in the TP53 transcript.

FIG. 25 shows a schematic representation of the selecteddisease-relevant cDNA containing G to A mutation from ClinVar data andthe corresponding 111-nt arRNA.

FIG. 26 shows correction of pathogenic mutations by an exemplary LEAPERmethod. A to I correction of disease-relevant G>A mutation from ClinVardata by the corresponding 111-nt arRNA, targeting clinical-relatedmutations from six pathogenic genes as indicated (FIG. 25 and the tablesof the sequences of arRNAs and control RNAs and disease-related cDNAsbelow). Data are presented as the mean±s.e.m. (n=3); unpaired two-sidedStudent's t-test, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, notsignificant.

FIGS. 27A-27C show RNA editing in multiple human primary cells byexemplary LEAPER methods. FIG. 27A, Quantification of the EGFPpositive(EGFP⁺) cells induced by LEAPER-mediated RNA editing. Human primarypulmonary fibroblasts and human primary bronchial epithelial cells weretransfected with Reporter-1, along with the 151-nt control RNA (CtrlRNA₁₅₁) or the 151-nt targeting arRNA (arRNA₁₅₁) followed by FACSanalysis. FIGS. 27B and 27C, Deep sequencing results showing the editingrate on PPIB transcripts in human primary pulmonary fibroblasts, humanprimary bronchial epithelial cells (b), and human primary T cells (c).Data in a, b and Untreated group (c) are presented as the mean±s.e.m.(n=3); data of Ctrl RNA₁₅₁ and arRNA₁₅₁ (c) are presented as themean±s.e.m. (n=2).

FIGS. 28A-28D show targeted editing by lentiviral transduction of arRNAand electroporation of synthesized arRNA oligonucleotides. FIG. 28A,Quantification of the EGFP⁺ cells. HEK293T cells stably expressing theRepoter-1 were infected with lentivirus expressing 151-nt of Ctrl RNA orthe targeting arRNA. FACS analyses were performed 2 days and 8 days postinfection. The ratios of EGFP⁺ cells were normalized by lentiviraltransduction efficiency (BFP⁺ ratios). FIG. 28B, Deep sequencing resultsshowing the editing rate on the PPIB transcripts upon lentiviraltransduction of 151-nt arRNAs into HEK293T cells. FIG. 28C, Schematic ofthe PPIB sequence and the corresponding 111-nt targeting arRNA. *(inred) represents nucleotide with 2′-O-methylation and phosphorothioatelinkage. FIG. 28D, Deep sequencing results showing the editing rate onthe PPIB transcripts upon electroporation of 111-nt synthetic arRNAoligonucleotides into human primary T cells.

FIGS. 29A-29E show restoration of α-L-iduronidase activity in Hurlersyndrome patient-derived primary fibroblast by an exemplary LEAPERmethod. FIG. 29A, Top, genetic information of pathogenic mutation inpatient-derived fibroblast GM06214; Medium, schematic of the IDUA maturemRNA sequence of GM06214 cells (Black) containing a homozygous TGG>TAGmutation in exon 9 of the IDUA gene (Trp402Ter), and the corresponding111-nt targeting arRNA₁₁₁-IDUA-V1 (Blue); Bottom, schematic of the IDUApre-mRNA sequence of GM06214 cells (Black) and the corresponding 111-nttargeting arRNA₁₁₁-IDUA-V2 (Blue). *(in red) represents nucleotides with2′-O-methylation and phosphorothioate linkage. FIG. 29B, Measuring thecatalytic activity of α-L-iduronidase with 4-methylumbelliferylα-L-iduronidase substrate at different time points. Data are presentedas the mean±s.e.m. (n=2). FIG. 29C, Deep sequencing results showing thetargeted editing rate on IDUA transcripts in GM06214 cells, 48 hourspost electroporation. FIG. 29D, Top, schematic of the IDUA transcriptsequence covered by the 111-nt arRNAs. The arrow indicates the targetedadenosine. All adenosines were marked in red. Bottom, a heatmap ofediting rate on adenosines covered by indicated arRNAs in the IDUAtranscript (marked in the bold frame in blue). e, Quantitative PCRshowing the expressions of type I interferon, interferon-stimulatedgenes, and pro-inflammatory genes upon arRNA or poly(I:C)electroporation. Data are presented as the mean (n=3).

FIGS. 30A-30C shows three versions of dual fluorescence reporters(Reporter-1, -2 and -3), mCherry and EGFP. FIG. 30A, structure ofReporter-1, FIG. 30B, structure of Reporter-2, and FIG. 30C, structureof Reporter-3.

FIG. 31 shows the structure of the pLenti-dCas13-ADAR1DD.

FIG. 32 shows the structure of the pLenti-MCS-mCherry backbone.

FIG. 33 shows the structure of the pLenti-arRNA-BFP backbone.

FIG. 34 shows the detected genotype of IDUA in GM06214 cells. A C1205G>A mutation was inthegenome.

FIG. 35 shows the test result of electrotransfection conditions ofcells.

FIG. 36 shows enzyme activity of IDUA and rate of desired mutation incells transfected with dRNAs designed to target IDUA pre-mRNA andmRNAusing electroporation, respectively.

FIGS. 37A-37B show the test using IDUA-reporter. FIG. 37A shows theconstruction of IDUA-reporter. FIG. 37B shows the editing efficiency ofdRNAs of different lengths (symmetric truncations) in 293T-IDUA-Reportercells using electroporation (293T cells with IDUA-reporter).

FIG. 38 shows the enzyme activity and editing efficiency determined atdifferent time points in GM06214 cells electrotransfected with dRNAs ofdifferent lengths (symmetric truncations).

FIGS. 39A-39B show the determined IDUA enzyme activity (FIG. 39A) and Ato G mutation rate (FIG. 39B) in cells transfected with different dRNAs(symmetrical truncations, 3′ terminal truncations and 5′ terminaltruncations) using Lipofectamine RNAiMAX.

FIGS. 40A-40B show the comparison of enzyme activities in GM06214 cellstransfected with dRNAs of different lengths using Lipofectamine RNAiMAX.In FIG. 40A, bases on the 3′ terminus of the dRNAs were reduced one byone from 55-c-25 to 55-c-10. In FIG. 40B, bases on the 3′ terminus ofthe dRNA were reduced one by one from 55-c-16 to 55-c-5.

FIG. 41 shows the comparison of enzyme activities in GM06214 cellstransfected with dRNAs of different lengths (the length of 3′ terminuswas fixed to 15 nt or 20 nt, while the length of the 5′ terminus wasgradually reduced) using Lipofectamine RNAiMAX.

FIG. 42 shows the comparison of enzyme activities in GM06214 cellstransfected with 3 groups of dRNAs using Lipofectamine RNAiMAX. For thedRNAs in each group, the distance from the targeting nucleotide to 5′end is different. This figure also shows the low editing efficiency ofdRNAs which are less than 60 nt.

FIGS. 43A-43B show the editing efficiency of 71 nt and 76 nt dRNAs withdifferent chemical modifications. FIG. 43A shows the editing efficiencyusing enzyme activities. FIG. 43B show the editing efficiency using theA to G rate.

FIG. 44 shows the comparison of enzyme activities in cells transfectedwith dRNAs in this invention and a preferable RNA for exogenous enzymeindependent RNA base editing in the prior art.

FIGS. 45A-45D show the RNA editing result of the mutation in USH2A model(c.11864 G>A, p.Trp3955*) using the chemically modified dRNAs of thisinvention. MFI and % GFP represent the editing efficiency. FIG. 45Ashows the construction of USH2A construction. FIG. 45B shows the editingefficiency of dRNAs with 3′ and 5′ termini of equal length. FIG. 45Cshows the editing efficiency of dRNAs with 3′ and 5′ termini ofdifferent lengths. FIG. 45D shows the relatively low editing efficiencyof dRNAs of less than 60 nucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides RNA editing methods (referred herein as“LEAPER” methods) and specially designed RNAs, referred herein asdeaminase-recruiting RNAs (“dRNAs”) or ADAR-recruiting RNAs (“arRNAs”),to edit target RNAs in a host cell. Without being bound by any theory orhypothesis, the dRNA acts through hybridizing to its target RNA in asequence-specific fashion to form a double-stranded RNA, which recruitsan Adenosine Deaminase Acting on RNA (ADAR) to deaminate a targetadenosine in the target RNA. As such, efficient RNA editing can beachieved in some embodiments without ectopic or overexpression of theADAR proteins in the host cell. Also provided are methods andcompositions for treating or preventing a disease or condition in anindividual using the RNA editing methods.

The RNA editing methods described herein do not use fusion proteinscomprising an ADAR and a protein that specifically binds to a guidenucleic acid, such as Cas. The deaminase-recruiting RNAs (“dRNA”)described herein do not comprise crRNA, tracrRNA or gRNA used in theCRISPR/Cas system. In some embodiments, the dRNA does not comprise anADAR-recruiting domain, or chemical modification(s). In someembodiments, the arRNA can be expressed from a plasmid or a viralvector, or synthesized as an oligonucleotide, which could achievedesirable editing efficiency. Without being bound by any theory orunderlying mechanism, it was discovered that certain dRNA with specificlength, location of the mismatch, and/or modification patterndemonstrate higher efficiency in RNA editing. The present applicationthus further provides improved RNA editing methods over those previouslyreported.

The LEAPER methods described herein have manageable off-target rates onthe targeted transcripts and rare global off-targets. Inventors haveused the LEAPER method to restore p53 function by repairing a specificcancer-relevant point mutation. The LEAPER methods described herein canalso be applied to a broad spectrum of cell types including multiplehuman primary cells, and can be used to restore the α-L-iduronidasecatalytic activity in Hurler syndrome patient-derived primaryfibroblasts without evoking innate immune responses. In someembodiments, the LEAPER method involves a single molecule (i.e., dRNA)system. The LEAPER methods described herein enable precise and efficientRNA editing, which offers transformative potential for basic researchand therapeutics.

Definitions

The present invention will be described with respect to particularembodiments and with reference to certain drawings but the invention isnot limited thereto but only by the claims. Any reference signs in theclaims shall not be construed as limiting the scope. Where the term“comprising” is used in the present description and claims, it does notexclude other elements or steps. Where an indefinite or definite articleis used when referring to a singular noun e.g. “a” or “an”, “the”, thisincludes a plural of that noun unless something else is specificallystated. For the recitation of numeric ranges of nucleotides herein, eachintervening number there between, is explicitly contemplated. Forexample, for the range of 40-260 nucleotides, any integer of nucleotidesbetween 40 and 260 nucleotides is contemplated in addition to thenumbers of 40 nucleotides and 260 nucleotides.

The following terms or definitions are provided solely to aid in theunderstanding of the invention. Unless specifically defined herein, allterms used herein have the same meaning as they would to one skilled inthe art of the present invention. Practitioners are particularlydirected to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Press, Plainsview, N.Y. (1989); and Ausubel etal., Current Protocols in Molecular Biology (Supplement 47), John Wiley& Sons, New York (1999), for definitions and terms of the art. Thedefinitions provided herein should not be construed to have a scope lessthan understood by a person of ordinary skill in the art.

The terms “deaminase-recruiting RNA,” “dRNA,” “ADAR-recruiting RNA” and“arRNA” are used herein interchangeably to refer to an engineered RNAcapable of recruiting an ADAR to deaminate a target adenosine in an RNA.

The terms “polynucleotide”, “nucleotide sequence” and “nucleic acid” areused interchangeably. They refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Two nucleotides are linked by a phosphodiester bond, andmultiple nucleotides are linked by phosphodiester bonds to formpolynucleotide or nucleic acid. The linkage between nucleotides can bephosphorothioated, called “phosphorothioate linkage” or“phosphorothioation linkage”.

The terms “adenine”, “guanine”, “cytosine”, “thymine”, “uracil” and“hypoxanthine” as used herein refer to the nucleobases as such. Theterms “adenosine”, “guanosine”, “cytidine”, “thymidine”, “uridine” and“inosine”, refer to the nucleobases linked to the ribose or deoxyribosesugar moiety. The term “nucleoside” refers to the nucleobase linked tothe ribose or deoxyribose. The term “nucleotide” refers to therespective nucleobase-ribosyl-phosphate ornucleobase-deoxyribosyl-phosphate. Sometimes the terms adenosine andadenine (with the abbreviation, “A”), guanosine and guanine (with theabbreviation, “G”), cytosine and cytidine (with the abbreviation, “C”),uracil and uridine (with the abbreviation, “U”), thymine and thymidine(with the abbreviation, “T”), inosine and hypo-xanthine (with theabbreviation, “I”), are used interchangeably to refer to thecorresponding nucleobase, nucleoside or nucleotide. Sometimes the termsnucleobase, nucleoside and nucleotide are used interchangeably, unlessthe context clearly requires differently.

In the context of the present application, “target RNA” refers to an RNAsequence to which a deaminase-recruiting RNA sequence is designed tohave perfect complementarity or substantial complementarity, andhybridization between the target sequence and the dRNA forms a doublestranded RNA (dsRNA) region containing a target adenosine, whichrecruits an adenosine deaminase acting on RNA (ADAR) that deaminates thetarget adenosine. In some embodiments, the ADAR is naturally present ina host cell, such as a eukaryotic cell (preferably, a mammalian cell,more preferably, a human cell). In some embodiments, the ADAR isintroduced into the host cell.

As used herein, “complementarity” refers to the ability of a nucleicacid to form hydrogen bond(s) with another nucleic acid by traditionalWatson-Crick base-pairing. A percent complementarity indicates thepercentage of residues in a nucleic acid molecule which can formhydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleicacid (e.g., about 5, 6, 7, 8, 9, 10 out of 10, being about 50%, 60%,70%, 80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence form hydrogen bonds with the same number of contiguous residuesin a second nucleic acid sequence. “Substantially complementary” as usedherein refers to a degree of complementarity that is at least about anyone of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over aregion of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology—Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. A sequence capable of hybridizingwith a given sequence is referred to as the “complement” of the givensequence.

As used herein, the terms “cell”, “cell line”, and “cell culture” areused interchangeably and all such designations include progeny. It isunderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as the original cellsare included.

Methods of RNA Editing

In this invention, the dRNA used herein comprises an RNA sequencecomprising a cytidine (C), adenosine (A) or uridine (U) directlyopposite the target adenosine to be edited in the target RNA whenbinding with the target RNA. The cytidine (C), adenosine (A) and uridine(U) directly opposite the target adenosine are collectively referred toas “targeting nucleotide”, or separately “targeting C”, “targeting A”,and “targeting U”. The targeting nucleotide and the two nucleotidesdirectly adjacent to targeting nucleotide forms a triplet which isherein referred to as “targeting triplet”.

In some embodiments, there is provided a method for editing a target RNAin a host cell (e.g., eukaryotic cell), comprising introducing adeaminase-recruiting RNA (dRNA) or a construct encoding the dRNA intothe host cell, wherein the dRNA comprises a complementary RNA sequencethat hybridizes to the target RNA, and wherein the dRNA is capable ofrecruiting an adenosine deaminase acting on RNA (ADAR) to deaminate atarget adenosine (A) in the target RNA.

In some embodiments, there is provided a method for editing a target RNAin a host cell (e.g., eukaryotic cell), comprising introducing a dRNA ora construct encoding the dRNA into the host cell, wherein the dRNAcomprises a complementary RNA sequence that hybridizes to the targetRNA, and wherein the dRNA recruits an endogenously expressed ADAR of thehost cell to deaminate a target A in the target RNA. In someembodiments, the method does not comprise introducing any protein orconstruct encoding a protein (e.g., Cas, ADAR or a fusion protein ofADAR and Cas) to the host cell.

In some embodiments, there is provided a method for editing a target RNAin a host cell (e.g., eukaryotic cell), comprising introducing: (a) adRNA or a construct encoding the dRNA, and (b) an ADAR or a constructencoding the ADAR into the host cell, wherein the dRNA comprises acomplementary RNA sequence that hybridizes to the target RNA, andwherein the dRNA recruits the ADAR to deaminate a target A in the targetRNA. In some embodiments, the ADAR is an endogenously encoded ADAR ofthe host cell, wherein introduction of the ADAR comprisesover-expressing the ADAR in the host cell. In some embodiments, the ADARis exogenous to the host cell. In some embodiments, the constructencoding the ADAR is a vector, such as a plasmid, or a viral vector(e.g., a lentiviral vector).

In some embodiments, there is provided a method for editing a plurality(e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100 or more) of targetRNAs in host cells (e.g., eukaryotic cells), comprising introducing aplurality of dRNAs or constructs encoding the plurality of dRNAs intothe host cell, wherein each dRNA comprises a complementary RNA sequencethat hybridizes to a corresponding target RNA in the plurality of targetRNAs, and wherein each dRNA is capable of recruiting an ADAR todeaminate a target A in the corresponding target RNA.

In some embodiments, there is provided a method for editing a plurality(e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100 or more) of targetRNAs in host cells (e.g., eukaryotic cells), comprising introducing aplurality of dRNAs or constructs encoding the plurality of dRNAs intothe host cell, wherein each dRNA comprises a complementary RNA sequencethat hybridizes to a corresponding target RNA in the plurality of targetRNAs, and wherein each dRNA recruits an endogenously expressed ADAR todeaminate a target A in the corresponding target RNA.

In some embodiments, there is provided a method for editing a plurality(e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100, 1000 or more) oftarget RNAs in host cells (e.g., eukaryotic cells), comprisingintroducing: (a) a plurality of dRNAs or constructs encoding theplurality of dRNAs, and (b) an ADAR or a construct encoding ADAR intothe host cells, wherein each dRNA comprises a complementary RNA sequencethat hybridizes to a corresponding target RNA in the plurality of targetRNAs, and wherein each dRNA recruits the ADAR to deaminate a target A inthe corresponding target RNA.

In one aspect, the present application provides a method for editing aplurality of RNAs in host cells by introducing a plurality of thedeaminase-recruiting RNAs, one or more constructs encoding thedeaminase-recruiting RNAs, or a library described herein, into the hostcells.

In certain embodiments, the method for editing on a target RNA comprisesintroducing multiple deaminase-recruiting RNAs or one or more constructscomprising the multiple deaminase-recruiting RNAs into host cells torecruit adenosine deaminase acting on RNA (ADAR) to perform deaminationreaction on one or more target adenosines in one or more target RNAs,wherein each deaminase-recruiting RNA comprises a RNA sequencescomplementary to a corresponding target RNA.

In one aspect, the present application provides a method for generatingone or more modifications in a target RNA and/or the protein encoded bya target RNA in a host cell (e.g., eukaryotic cell), comprisingintroducing a dRNA or a construct encoding the dRNA into the host cell,wherein the dRNA comprises a complementary RNA sequence that hybridizesto the target RNA, and wherein the one or more modifications areselected from the group consisting of a point mutation of the proteinencoded by the target RNA, misfolding of the protein encoded by thetarget RNA, an early stop codon in the target RNA, an aberrant splicesite in the target RNA, and an alternative splice site in the targetRNA.

In certain embodiments, the method for generating one or moremodifications in a target RNA and/or the protein encoded by a target RNAin host cells (e.g., eukaryotic cells), comprises introducing aplurality of deaminase-recruiting RNAs or constructs encoding theplurality of deaminase-recruiting RNAs into the host cells, wherein eachdRNA comprises a complementary RNA sequence that hybridizes to acorresponding target RNA in the plurality of target RNAs, and whereineach dRNA is capable of recruiting an ADAR to deaminate a target A inthe corresponding target RNA.

In one aspect, the present application provides use of adeaminase-recruiting RNA according to any one of the dRNAs describedherein for editing a target RNA in a host cell. In certain embodiments,the deaminase-recruiting RNA comprises a complementary RNA sequence thathybridizes to the target RNA to be edited.

In one aspect, the present application provides use of adeaminase-recruiting RNA according to any one of the dRNAs describedherein for generating one or more modifications on a target RNA and/orthe protein encoded by a target RNA, wherein the one or moremodifications are selected from a group consisting of a point mutationof the protein encoded by the target RNA, misfolding of the proteinencoded by the target RNA, an early stop codon in the target RNA, anaberrant splice site in the target RNA, and an alternative splice sitein the target RNA. In certain embodiments, the deaminase-recruiting RNAcomprises a complementary RNA sequence that hybridizes to the target RNAto be edited.

The invention also relates to a method for leveraging an endogenousadenosine deaminase for editing a target RNA in a eukaryotic cell,comprising introducing a dRNA or a construct encoding the dRNA, asdescribed herein, into the eukaryotic cell to recruit naturallyendogenous adenosine deaminase acting on RNA (ADAR) to performdeamination reaction on a target adenosine in the target RNA sequence.

In certain embodiments according to any one of the methods or usedescribed herein, the dRNA comprises at least about any one of 40, 45,50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, or 250 nucleotides. In certainembodiments, the dRNA is about any one of 40-260, 45-250, 50-240,60-230, 65-220, 70-220, 70-210, 70-200, 70-190, 70-180, 70-170, 70-160,70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 75-200,80-190, 85-180, 90-170, 95-160, 100-200, 100-150, 100-175, 110-200,110-175, 110-150, or 105-140 nucleotides in length. In some embodimentsthe dRNA is about 60-200, such as about any of 60-150, 65-140, 68-130,or 70-120) nucleotides long. In some embodiments, the dRNA is about 71nucleotides long. In some embodiments, the dRNA is about 111 nucleotideslong.

In certain embodiments according to any one of the methods or usedescribed herein, the dRNA does not comprise an ADAR-recruiting domain.“ADAR-recruiting domain” can be a nucleotide sequence or structure thatbinds at high affinity to ADAR, or a nucleotide sequence that binds to abinding partner fused to ADAR in an engineered ADAR construct. ExemplaryADAR-recruiting domains include, but are not limited to, GluR-2, GluR-B(R/G), GluR-B (Q/R), GluR-6 (R/G), 5HT2C, and FlnA (Q/R) domain; see,for example, Wahlstedt, Helene, and Marie, “Site-selective versuspromiscuous A-to-I editing.” Wiley Interdisciplinary Reviews: RNA 2.6(2011): 761-771, which is incorporated herein by reference in itsentirety. In some embodiments, the dRNA does not comprise adouble-stranded portion. In some embodiments, the dRNA does not comprisea hairpin, such as MS2 stem loop. In some embodiments, the dRNA issingle stranded. In some embodiments, the dRNA does not comprise aDSB-binding domain. In some embodiments, the dRNA consists of (orconsists essentially of) the complementary RNA sequence.

In certain embodiments according to any one of the methods or usedescribed herein, the dRNA does not comprise chemical modifications. Insome embodiments, the dRNA does not comprise a chemically modifiednucleotide, such as 2′-O-methyl nucleotide or a nucleotide having aphosphorothioate linkage. In some embodiments, the dRNA comprises2′-O-methylation and phosphorothioate linkage only at the first threeand last three residues. In some embodiments, the dRNA is not anantisense oligonucleotide (ASO).

In certain embodiments according to any one of the methods or usedescribed herein, the host cell is a prokaryotic cell. In someembodiments, the host cell is a eukaryotic cell. Preferably, the hostcell is a mammalian cell. Most preferably, the host cell is a humancell. In some embodiments, the host cell is a murine cell. In someembodiments, the host cell is a plant cell or a fungal cell.

In some embodiments according to any one of the methods or use describedherein, the host cell is a cell line, such as HEK293T, HT29, A549,HepG2, RD, SF268, SW13 and HeLa cell. In some embodiments, the host cellis a primary cell, such as fibroblast, epithelial, or immune cell. Insome embodiments, the host cell is a T cell. In some embodiments, thehost cell is a post-mitosis cell. In some embodiments, the host cell isa cell of the central nervous system (CNS), such as a brain cell, e.g.,a cerebellum cell.

In some embodiments, there is provided a method of editing a target RNAin a primary host cell (e.g., T cell or a CNS cell) comprisingintroducing a dRNA or a construct encoding the dRNA into the host cell,wherein the dRNA comprises a complementary RNA sequence that hybridizesto the target RNA, and wherein the dRNA recruits an endogenouslyexpressed ADAR of the host cell to deaminate a target A in the targetRNA.

In certain embodiments according to any one of the methods or usedescribed herein, the ADAR is endogenous to the host cell. In someembodiments, the adenosine deaminase acting on RNA (ADAR) is naturallyor endogenously present in the host cell, for example, naturally orendogenously present in the eukaryotic cell. In some embodiments, theADAR is endogenously expressed by the host cell. In certain embodiments,the ADAR is exogenously introduced into the host cell. In someembodiments, the ADAR is ADAR1 and/or ADAR2. In certain embodiments, theADAR is one or more ADARs selected from the group consisting of hADAR1,hADAR2, mouse ADAR1 and ADAR2. In some embodiments, the ADAR is ADAR1,such as p110 isoform of ADAR1 (“ADAR1^(p110)”) and/or p150 isoform ofADAR1 (“ADAR^(p150)”). In some embodiments, the ADAR is ADAR2. In someembodiments, the ADAR is an ADAR2 expressed by the host cell, e.g.,ADAR2 expressed by cerebellum cells.

In some embodiments, the ADAR is an ADAR exogenous to the host cell. Insome embodiments, the ADAR is a hyperactive mutant of a naturallyoccurring ADAR. In some embodiments, the ADAR is ADAR1 comprising anE1008Q mutation. In some embodiments, the ADAR is not a fusion proteincomprising a binding domain. In some embodiments, the ADAR does notcomprise an engineered double-strand nucleic acid-binding domain. Insome embodiments, the ADAR does not comprise a MCP domain that binds toMS2 hairpin that is fused to the complementary RNA sequence in the dRNA.In some embodiments, the ADAR does not comprise a DSB.

In some embodiments according to any one of the methods or use describedherein, the host cell has high expression level of ADAR1 (such asADAR1^(p110) and/or ADAR1^(p150)), e.g., at least about any one of 10%,20%, 50%, 100%, 2×, 3×, 5×, or more relative to the protein expressionlevel of β-tubulin. In some embodiments, the host cell has highexpression level of ADAR2, e.g., at least about any one of 10%, 20%,50%, 100%, 2×, 3×, 5×, or more relative to the protein expression levelof β-tubulin. In some embodiments, the host cell has low expressionlevel of ADAR3, e.g., no more than about any one of 5×, 3×, 2×, 100%,50%, 20% or less relative to the protein expression level of β-tubulin.

In certain embodiments according to any one of the methods or usedescribed herein, the complementary RNA sequence comprises a cytidine,adenosine or uridine directly opposite the target A in the target RNA.In some embodiments, complementary RNA sequence comprises a cytidinemismatch directly opposite the target A in the target RNA. In someembodiments, the cytidine mismatch is located at least 5 nucleotides,e.g., at least 10, 15, 20, 25, 30, or more nucleotides, away from the 5′end of the complementary RNA sequence. In some embodiments, the cytidinemismatch is located at least 20 nucleotides, e.g., at least 25, 30, 35,or more nucleotides, away from the 3′ end of the complementary RNAsequence. In some embodiments, the cytidine mismatch is not locatedwithin 20 (e.g., 15, 10, 5 or fewer) nucleotides away from the 3′ end ofthe complementary RNA sequence. In some embodiments, the cytidinemismatch is located at least 20 nucleotides (e.g., at least 25, 30, 35,or more nucleotides) away from the 3′ end and at least 5 nucleotides(e.g., at least 10, 15, 20, 25, 30, or more nucleotides) away from the5′ end of the complementary RNA sequence. In some embodiments, thecytidine mismatch is located in the center of the complementary RNAsequence. In some embodiments, the cytidine mismatch is located within20 nucleotides (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide)of the center of the complementary sequence in the dRNA.

The dRNA described herein can also be characterized as comprising, from5′ end to 3′ end: a 5′ portion, a cytidine mismatch directly opposite tothe target A in the target RNA, and a 3′ portion. In some embodiments,the 3′ portion is no shorter than about 7 nt (such as no shorter than 8nt, no shorter than 9 nt, and no shorter than 10 nt) nucleotides. Insome embodiments, the 3′ portion is about 7 nt-25 nt nucleotide long(such as about 10 nt-15 nt or 21 nt-25 nt nucleotides long). In someembodiments, the 5′ portion is no shorter than about 25 (such as noshorter than about 30, no shorter than about 35 nt, no shorter thanabout 40 nt, and no shorter than about 45 nt) nucleotides. In someembodiments, the 5′ portion is about 25 nt-85 nt nucleotides long (suchas about 25 nt-80 nt, 25 nt-75 nt, 25 nt-70 nt, 25 nt-65 nt, 25 nt-60nt, 30 nt-55 nt, 40 nt-55 nt, or 45 nt-55 nt nucleotides long). In someembodiments, the 5′ portion is about 25 nt-85 nt nucleotides long (suchas about 25 nt-80 nt, 25 nt-75 nt, 25 nt-70 nt, 25 nt-65 nt, 25 nt-60nt, 30 nt-55 nt, 40 nt-55 nt, or 45 nt-55 nt nucleotides long), and the3′ portion is about 7 nt-25 nt nucleotide long (such as about 10 nt-15nt or 21 nt-25 nt nucleotides long). In some embodiments, the 5′ portionis longer than the 3′ portion. In some embodiments, the 5′ portion isabout 55 nucleotides long, and the 3′ portion is about 15 nucleotideslong.

In some embodiments, the position of the cytidine mismatch in the dRNAis according to any of the dRNAs described in the examples herein, andthe dRNA can be, for example, in the format of Xnt-c-Ynt, wherein Xrepresents the length of the 5′ portion and Y represents the length ofthe 3′ portion: 55 nt-c-35 nt, 55 nt-c-25 nt, 55 nt-c-24 nt, 55 nt-c-23nt, 55 nt-c-22 nt, 55 nt-c-21 nt, 55 nt-c-20 nt, 55 nt-c-19 nt, 55nt-c-18 nt, 55 nt-c-17 nt, 55 nt-c-16 nt, 55 nt-c-15 nt, 55 nt-c-14 nt,55 nt-c-13 nt, 55 nt-c-12 nt, 55 nt-c-11 nt, 55 nt-c-10 nt, 55 nt-c-9nt, 55 nt-c-8 nt, 55 nt-c-7 nt, 55 nt-n-20 nt, 50 nt-n-20 nt, 45 nt-n-20nt, 55 nt-n-15 nt, 50 nt-n-15 nt, 45 nt-c-45 nt, 45 nt-c-55 nt, 54nt-c-12 nt, 53 nt-c-13 nt, 52 nt-c-14 nt, 51 nt-c-15 nt, 50 nt-c-16 nt,49 nt-c-17 nt, 48 nt-c-18 nt, 47 nt-c-19 nt, 46 nt-c-20 nt, 45 nt-c-21nt, 44 nt-c-22 nt, 43 nt-c-23 nt, 54 nt-c-15 nt, 53 nt-c-16 nt, 52nt-c-17 nt, 51 nt-c-18 nt, 50 nt-c-19 nt, 49 nt-c-20 nt, 48 nt-c-21 nt,47 nt-c-22 nt, 46 nt-c-23 nt, 54 nt-c-17 nt, 53 nt-n-18 nt, 52 nt-n-19nt, 51 nt-n-20 nt, 50 nt-n-21 nt, 49 nt-n-22 nt, 48 nt-c-23.

In certain embodiments according to any one of the methods or usedescribed herein, the complementary RNA sequence further comprises oneor more guanosine(s), such as 1, 2, 3, 4, 5, 6, or more Gs, that is eachdirectly opposite a non-target adenosine in the target RNA. In someembodiments, the complementary RNA sequence comprises two or moreconsecutive mismatch nucleotides (e.g., 2, 3, 4, 5, or more mismatchnucleotides) opposite a non-target adenosine in the target RNA. In someembodiments, the target RNA comprises no more than about 20 non-targetAs, such as no more than about any one of 15, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 non-target A. The Gs and consecutive mismatch nucleotidesopposite non-target As may reduce off-target editing effects by ADAR.

In certain embodiments according to any one of the methods or usedescribed herein, the 5′ nearest neighbor of the target A is anucleotide selected from U, C, A and G with the preference U>C≈A>G andthe 3′ nearest neighbor of the target A is a nucleotide selected from G,C, A and U with the preference G>C>A≈U. In certain embodiments, thetarget A is in a three-base motif selected from the group consisting ofUAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC,GAA and GAU in the target RNA. In certain embodiments, the three-basemotif is UAQ and the dRNA comprises an A directly opposite the U in thethree-base motif, a C directly opposite the target A, and a C, G or Udirectly opposite the G in the three-base motif. In certain embodiments,the three-base motif is UAG in the target RNA, and the dRNA comprisesACC, ACG or ACU that is opposite the UAG of the target RNA. In certainembodiments, the three-base motif is UAG in the target RNA, and the dRNAcomprises ACC that is opposite the UAG of the target RNA.

In some embodiments, the dRNA comprises one or more modifications.Exemplary modifications to the dRNA include, but are not limited to,phosphorothioate backbone modification, 2′-substitutions in the ribose(such as 2′-O-methylation and 2′-fluoro substitutions), LNA, and L-RNA.In some embodiments, the dRNA comprises one or more modifications, suchas 2′-O-methylation and/or phosphorothioation. In some embodiments, thedRNA is of about 60-200 (This range covers any consecutive positiveintegers between the numbers 60 and 200, for example, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180,190, 200) nucleotides long and comprises one or more moficiations (suchas 2′-O-methylation and/or 3′-phosphorothioation). In some embodiments,the dRNA is of about 60-200 nucleotides long and comprises one or moremoficiations. In some embodiments, the dRNA is of about 60-200nucleotides long and comprises 2′-O-methylation and/orphosphorothioation moficiations. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides and/orphosphorothiations in the first and last 3 internucleotide linkages. Insome embodiments, the dRNA comprises 2′-O-methylations in the first andlast 3 nucleotides, phosphorothiations in the first and last 3internucleotide linkages, and 2′-O-methylations in one or more uridines,for example on all uridines. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in a single or multiple or all uridines, and amodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine. In certain embodiments, the modification in thenucleotide opposite to the target adenosine, and/or one or twonucleotides most adjacent to the nucleotide opposite to the targetadenosine is a 2′-O-methylation. In certain embodiments, themodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine is a phosphorothiation linkage, such as a3′-phosphorothiation linkage. In certain embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in all uridines, and a 2′-O-methylation in thenucleotide adjacent to the 3′ terminus and/or 5′ terminus of thenucleotide opposite to the target adenosine. In certain embodiments, thedRNA comprises 2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in a single or multiple or all uridines, and aphosphorothiation linkage such as 3′-phosphorothiation linkage in thenucleotide opposite to the target adenosine and/or its 5′ and/or 3′ mostadjacent nucleotides. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 5 nucleotides andphosphorothiations in the first and last 5 internucleotide linkages.

In certain embodiments according to any one of the methods or usedescribed herein, the target RNA is any one selected from the groupconsisting of a pre-messenger RNA, a messenger RNA, a ribosomal RNA, atransfer RNA, a long non-coding RNA and a small RNA (e.g., miRNA). Insome embodiments, the target RNA is a pre-messenger RNA. In someembodiments, the target RNA is a messenger RNA.

In certain embodiments according to any one of the methods or usedescribed herein, the method further comprises introducing an inhibitorof ADAR3 to the host cell. In some embodiments, the inhibitor of ADAR3is an RNAi against ADAR3, such as a shRNA against ADAR3 or a siRNAagainst ADAR3. In some embodiments, the method further comprisesintroducing a stimulator of interferon to the host cell. In someembodiments, the ADAR is inducible by interferon, for example, the ADARis ADAR^(p150). In some embodiments, the stimulator of interferon isIFNα. In some embodiments, the inhibitor of ADAR3 and/or the stimulatorof interferon are encoded by the same construct (e.g., vector) thatencodes the dRNA.

In certain embodiments according to any one of the methods or usedescribed herein, the efficiency of editing of the target RNA is atleast about 20%, such as at least about any one of 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or higher. In someembodiments, the efficiency of editing is determined by Sangersequencing. In some embodiments, the efficiency of editing is determinedby next-generation sequencing.

In certain embodiments according to any one of the methods or usedescribed herein, the method has low off-target editing rate. In someembodiments, the method has lower than about 1% (e.g., no more thanabout any one of 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or lower) editingefficiency on non-target As in the target RNA. In some embodiments, themethod does not edit non-target As in the target RNA. In someembodiments, the method has lower than about 0.1% (e.g., no more thanabout any one of 0.05%, 0.01%, 0.005%, 0.001%, 0.0001% or lower) editingefficiency on As in non-target RNA.

In certain embodiments according to any one of the methods or usedescribed herein, the method does not induce immune response, such asinnate immune response. In some embodiments, the method does not induceinterferon and/or interleukin expression in the host cell. In someembodiments, the method does not induce IFN-β and/or IL-6 expression inthe host cell.

Also provided are edited RNA or host cells having an edited RNA producedby any one of the methods described herein. In some embodiments, theedited RNA comprises an inosine. In some embodiments, the host cellcomprises an RNA having a missense mutation, an early stop codon, analternative splice site, or an aberrant splice site. In someembodiments, the host cell comprises a mutant, truncated, or misfoldedprotein.

“Host cell” as described herein refers to any cell type that can be usedas a host cell provided it can be modified as described herein. Forexample, the host cell may be a host cell with endogenously expressedadenosine deaminase acting on RNA (ADAR), or may be a host cell intowhich an adenosine deaminase acting on RNA (ADAR) is introduced by aknown method in the art. For example, the host cell may be a prokaryoticcell, a eukaryotic cell or a plant cell. In some embodiments, the hostcell is derived from a pre-established cell line, such as mammalian celllines including human cell lines or non-human cell lines. In someembodiments, the host cell is derived from an individual, such as ahuman individual.

“Introducing” or “introduction” used herein means delivering one or morepolynucleotides, such as dRNAs or one or more constructs includingvectors as described herein, one or more transcripts thereof, to a hostcell. The invention serves as a basic platform for enabling targetedediting of RNA, for example, pre-messenger RNA, a messenger RNA, aribosomal RNA, a transfer RNA, a long non-coding RNA and a small RNA(such as miRNA). The methods of the present application can employ manydelivery systems, including but not limited to, viral, liposome,electroporation, microinjection and conjugation, to achieve theintroduction of the dRNA or construct as described herein into a hostcell. Conventional viral and non-viral based gene transfer methods canbe used to introduce nucleic acids into mammalian cells or targettissues. Such methods can be used to administer nucleic acids encodingdRNA of the present application to cells in culture, or in a hostorganism. Non-viral vector delivery systems include DNA plasmids, RNA(e.g. a transcript of a construct described herein), naked nucleic acid,and nucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes for delivery to the host cell.

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid: nucleic acid conjugates,electroporation, nanoparticles, exosomes, microvesicles, or gene-gun,naked DNA and artificial virions.

The use of RNA or DNA viral based systems for the delivery of nucleicacids has high efficiency in targeting a virus to specific cells andtrafficking the viral payload to the cellular nuclei.

In certain embodiments according to any one of the methods or usedescribed herein, the method comprises introducing a viral vector (suchas lentiviral vector) encoding the dRNA to the host cell. In someembodiments, the method comprises introducing a plasmid encoding thedRNA to the host cell. In some embodiments, the method comprisesintroducing (e.g., by electroporation) the dRNA (e.g., synthetic dRNA)into the host cell. In some embodiments, the method comprisestransfection of the dRNA into the host cell.

After deamination, modification of the target RNA and/or the proteinencoded by the target RNA, can be determined using different methodsdepending on the positions of the targeted adenosines in the target RNA.For example, in order to determine whether “A” has been edited to “I” inthe target RNA, RNA sequencing methods known in the art can be used todetect the modification of the RNA sequence. When the target adenosineis located in the coding region of an mRNA, the RNA editing may causechanges to the amino acid sequence encoded by the mRNA. For example,point mutations may be introduced to the mRNA of an innate or acquiredpoint mutation in the mRNA may be reversed to yield wild-type geneproduct(s) because of the conversion of “A” to “I”. Amino acidsequencing by methods known in the art can be used to find any changesof amino acid residues in the encoded protein. Modifications of a stopcodon may be determined by assessing the presence of a functional,elongated, truncated, full-length and/or wild-type protein. For example,when the target adenosine is located in a UGA, UAQ or UAA stop codon,modification of the target A (UGA or UAG) or As (UAA) may create aread-through mutation and/or an elongated protein, or a truncatedprotein encoded by the target RNA may be reversed to create afunctional, full-length and/or wild-type protein. Editing of a targetRNA may also generate an aberrant splice site, and/or alternative splicesite in the target RNA, thus leading to an elongated, truncated, ormisfolded protein, or an aberrant splicing or alternative splicing siteencoded in the target RNA may be reversed to create a functional,correctly-folding, full-length and/or wild-type protein. In someembodiments, the present application contemplates editing of both innateand acquired genetic changes, for example, missense mutation, early stopcodon, aberrant splicing or alternative splicing site encoded by atarget RNA. Using known methods to assess the function of the proteinencoded by the target RNA can find out whether the RNA editing achievesthe desired effects. Because deamination of the adenosine (A) to aninosine (I) may correct a mutated A at the target position in a mutantRNA encoding a protein, identification of the deamination into inosinemay provide assessment on whether a functional protein is present, orwhether a disease or drug resistance-associated RNA caused by thepresence of a mutated adenosine is reversed or partly reversed.Similarly, because deamination of the adenosine (A) to an inosine (I)may introduce a point mutation in the resulting protein, identificationof the deamination into inosine may provide a functional indication foridentifying a cause of disease or a relevant factor of a disease.

When the presence of a target adenosine causes aberrant splicing, theread-out may be the assessment of occurrence and frequency of aberrantsplicing. On the other hand, when the deamination of a target adenosineis desirable to introduce a splice site, then similar approaches can beused to check whether the required type of splicing occurs. An exemplarysuitable method to identify the presence of an inosine after deaminationof the target adenosine is RT-PCR and sequencing, using methods that arewell-known to the person skilled in the art.

The effects of deamination of target adenosine(s) include, for example,point mutation, early stop codon, aberrant splice site, alternativesplice site and misfolding of the resulting protein. These effects mayinduce structural and functional changes of RNAs and/or proteinsassociated with diseases, whether they are genetically inherited orcaused by acquired genetic mutations, or may induce structural andfunctional changes of RNAs and/or proteins associated with occurrence ofdrug resistance. Hence, the dRNAs, the constructs encoding the dRNAs,and the RNA editing methods of present application can be used inprevention or treatment of hereditary genetic diseases or conditions, ordiseases or conditions associated with acquired genetic mutations bychanging the structure and/or function of the disease-associated RNAsand/or proteins.

In some embodiments, the target RNA is a regulatory RNA. In someembodiments, the target RNA to be edited is a ribosomal RNA, a transferRNA, a long non-coding RNA or a small RNA (e.g., miRNA, pri-miRNA,pre-miRNA, piRNA, siRNA, snoRNA, snRNA, exRNA or scaRNA). The effects ofdeamination of the target adenosines include, for example, structuraland functional changes of the ribosomal RNA, transfer RNA, longnon-coding RNA or small RNA (e.g., miRNA), including changes ofthree-dimensional structure and/or loss of function or gain of functionof the target RNA. In some embodiments, deamination of the target As inthe target RNA changes the expression level of one or more downstreammolecules (e.g., protein, RNA and/or metabolites) of the target RNA.Changes of the expression level of the downstream molecules can beincrease or decrease in the expression level.

Some embodiments of the present application involve multiplex editing oftarget RNAs in host cells, which are useful for screening differentvariants of a target gene or different genes in the host cells. In someembodiments, wherein the method comprises introducing a plurality ofdRNAs to the host cells, at least two of the dRNAs of the plurality ofdRNAs have different sequences and/or have different target RNAs. Insome embodiments, each dRNA has a different sequence and/or differenttarget RNA. In some embodiments, the method generates a plurality (e.g.,at least 2, 3, 5, 10, 50, 100, 1000 or more) of modifications in asingle target RNA in the host cells. In some embodiments, the methodgenerates a modification in a plurality (e.g., at least 2, 3, 5, 10, 50,100, 1000 or more) of target RNAs in the host cells. In someembodiments, the method comprises editing a plurality of target RNAs ina plurality of populations of host cells. In some embodiments, eachpopulation of host cells receive a different dRNA or a dRNAs having adifferent target RNA from the other populations of host cells.

Deaminase-Recruiting RNA, Construct, and Library

In one aspect, the present application provides a deaminase-recruitingRNA useful for any one of the methods described herein. Any one of thedRNAs described in this section may be used in the methods of RNAediting and treatment described herein. It is intended that any of thefeatures and parameters described herein for dRNAs can be combined witheach other, as if each and every combination is individually described.The dRNAs described herein do not comprise a tracrRNA, crRNA or gRNAused in a CRISPR/Cas system.

In some embodiments, there is provided a deaminase-recruiting RNA (dRNA)for deamination of a target adenosine in a target RNA by recruiting anADAR, comprising a complementary RNA sequence that hybridizes to thetarget RNA.

In one aspect, the present provides a construct comprising any one ofthe deaminase-recruiting RNAs described herein. In certain embodiments,the construct is a viral vector (preferably a lentivirus vector) or aplasmid. In some embodiments, the construct encodes a single dRNA. Insome embodiments, the construct encodes a plurality (e.g., about any oneof 1, 2, 3, 4, 5, 10, 20 or more) dRNAs.

In one aspect, the present application provides a library comprising aplurality of the deaminase-recruiting RNAs or a plurality of theconstructs described herein.

In one aspect, the present application provides a composition or a hostcell comprising the deaminase-recruiting RNA or the construct describedherein. In certain embodiments, the host cell is a prokaryotic cell or aeukaryotic cell. Preferably, the host cell is a mammalian cell. Mostpreferably, the host cell is a human cell.

In certain embodiments according to any one of the dRNAs, constructs,libraries or compositions described herein, the complementary RNAsequence comprises a cytidine, adenosine or uridine directly oppositethe target adenosine to be edited in the target RNA. In certainembodiments, the complementary RNA sequence further comprises one ormore guanosine(s) that is each directly opposite a non-target adenosinein the target RNA. In certain embodiments, the 5′ nearest neighbor ofthe target A is a nucleotide selected from U, C, A and G with thepreference U>C≈A>G and the 3′ nearest neighbor of the target A is anucleotide selected from Q C, A and U with the preference G>C>A≈U. Insome embodiments, the 5′ nearest neighbor of the target A is U. In someembodiments, the 5′ nearest neighbor of the target A is C or A. In someembodiments, the 3′ nearest neighbor of the target A is G In someembodiments, the 3′ nearest neighbor of the target A is C.

In certain embodiments according to any one of the dRNAs, constructs,libraries or compositions described herein, the target A is in athree-base motif selected from the group consisting of UAQ UAC, UAA,UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU inthe target RNA. In certain embodiments, the three-base motif is UAQ andthe dRNA comprises an A directly opposite the U in the three-base motif,a C directly opposite the target A, and a C, G or U directly oppositethe G in the three-base motif. In certain embodiments, the three-basemotif is UAG in the target RNA, and the dRNA comprises ACC, ACG or ACUthat is opposite the UAG of the target RNA.

In some embodiments, the dRNA comprises a cytidine mismatch directlyopposite the target A in the target RNA. In some embodiments, thecytidine mismatch is close to the center of the complementary RNAsequence, such as within 20, 15, 10, 5, 4, 3, 2, or 1 nucleotide awayfrom the center of the complementary RNA sequence. In some embodiments,the cytidine mismatch is at least 5 nucleotides away from the 5′ end ofthe complementary RNA sequence. In some embodiments, the cytidinemismatch is at least 20 nucleotides away from the 3′ end of thecomplementary RNA sequence.

In certain embodiments according to any one of the dRNAs, constructs,libraries or compositions described herein, the dRNA comprises at leastabout any one of 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250nucleotides. In certain embodiments, the dRNA is about any one of40-260, 45-250, 50-240, 60-230, 65-220, 70-210, 70-200, 70-190, 70-180,70-170, 70-160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90,70-80, 75-200, 80-190, 85-180, 90-170, 95-160, 100-150 or 105-140nucleotides in length. In some embodiments the dRNA is about 60-200(such as about any of 60-150, 65-140, 68-130, or 70-120) nucleotideslong.

The dRNA of the present application comprises a complementary RNAsequence that hybridizes to the target RNA. The complementary RNAsequence is perfectly complementary or substantially complementarity tothe target RNA to allow hybridization of the complementary RNA sequenceto the target RNA. In some embodiments, the complementary RNA sequencehas 100% sequence complementarity as the target RNA. In someembodiments, the complementary RNA sequence is at least about any one of70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more complementary toover a continuous stretch of at least about any one of 20, 40, 60, 80,100, 150, 200, or more nucleotides in the target RNA. In someembodiments, the dsRNA formed by hybridization between the complementaryRNA sequence and the target RNA has one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more) non-Watson-Crick base pairs (i.e., mismatches).

ADAR, for example, human ADAR enzymes edit double stranded RNA (dsRNA)structures with varying specificity, depending on a number of factors.One important factor is the degree of complementarity of the two strandsmaking up the dsRNA sequence. Perfect complementarity of between thedRNA and the target RNA usually causes the catalytic domain of ADAR todeaminate adenosines in a non-discriminative manner. The specificity andefficiency of ADAR can be modified by introducing mismatches in thedsRNA region. For example, A-C mismatch is preferably recommended toincrease the specificity and efficiency of deamination of the adenosineto be edited. Conversely, at the other A (adenosine) positions than thetarget A (i.e., “non-target A”), the G-A mismatch can reduce off-targetediting. Perfect complementarity is not necessarily required for a dsRNAformation between the dRNA and its target RNA, provided there issubstantial complementarity for hybridization and formation of the dsRNAbetween the dRNA and the target RNA. In some embodiments, the dRNAsequence or single-stranded RNA region thereof has at least about anyone of 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of sequencecomplementarity to the target RNA, when optimally aligned. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting examples of which include theSmith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g. the Burrows WheelerAligner).

The nucleotides neighboring the target adenosine also affect thespecificity and efficiency of deamination. For example, the 5′ nearestneighbor of the target adenosine to be edited in the target RNA sequencehas the preference U>C≈A>G and the 3′ nearest neighbor of the targetadenosine to be edited in the target RNA sequence has the preferenceG>C>A≈U in terms of specificity and efficiency of deamination ofadenosine. In some embodiments, when the target adenosine may be in athree-base motif selected from the group consisting of UAQ UAC, UAA,UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU inthe target RNA, the specificity and efficiency of deamination ofadenosine are higher than adenosines in other three-base motifs. In someembodiments, where the target adenosine to be edited is in thethree-base motif UAG, UAC, UAA, UAU, CAG, CAC, AAG, AAC or AAA, theefficiency of deamination of adenosine is much higher than adenosines inother motifs. With respect to the same three-base motif, differentdesigns of dRNA may also lead to different deamination efficiency.Taking the three-base motif UAG as an example, in some embodiments, whenthe dRNA comprises cytidine (C) directly opposite the target adenosineto be edited, adenosine (A) directly opposite the uridine, and cytidine(C), guanosine (G) or uridine (U) directly opposite the guanosine, theefficiency of deamination of the target adenosine is higher than thatusing other dRNA sequences. In some embodiments, when the dRNA comprisesACC, ACG or ACU opposite UAG of the target RNA, the editing efficiencyof the A in the UAG of the target RNA may reach about 25%-30%.

Besides the target adenosines, there may be one or more adenosines inthe target RNA which are not desirable to be edited. With respect tothese adenosines, it is preferable to reduce their editing efficiency asmuch as possible. It is found by this invention that where guanosine isdirectly opposite an adenosine in the target RNA, the deaminationefficiency is significantly decreased. Therefore, in order to decreaseoff-target deamination, dRNAs can be designed to comprise one or moreguanosines directly opposite one or more adenosine(s) other than thetarget adenosine to be edited in the target RNA.

The desired level of specificity and efficiency of editing the targetRNA sequence may depend on different applications. Following theinstructions in the present patent application, those of skill in theart will be capable of designing a dRNA having complementary orsubstantially complementary sequence to the target RNA sequenceaccording to their needs, and, with some trial and error, obtain theirdesired results. As used herein, the term “mismatch” refers to opposingnucleotides in a double stranded RNA (dsRNA) which do not form perfectbase pairs according to the Watson-Crick base pairing rules. Mismatchbase pairs include, for example, G-A, C-A, U-C, A-A, G-G, C-C, U-U basepairs. Taking A-C match as an example, where a target A is to be editedin the target RNA, a dRNA is designed to comprise a C opposite the A tobe edited, generating a A-C mismatch in the dsRNA formed byhybridization between the target RNA and dRNA.

In some embodiments, the dsRNA formed by hybridization between the dRNAand the target RNA does not comprise a mismatch. In some embodiments,the dsRNA formed by hybridization between the dRNA and the target RNAcomprises one or more, such as any one of 1, 2, 3, 4, 5, 6, 7 or moremismatches (e.g., the same type of different types of mismatches). Insome embodiments, the dsRNA formed by hybridization between the dRNA andthe target RNA comprises one or more kinds of mismatches, for example,1, 2, 3, 4, 5, 6, 7 kinds of mismatches selected from the groupconsisting of G-A, C-A, U-C, A-A, G-Q C-C and U-U.

The mismatch nucleotides in the dsRNA formed by hybridization betweenthe dRNA and the target RNA can form bulges which can promote theefficiency of editing of the target RNA. There may be one (which is onlyformed at the target adenosine) or more bulges formed by the mismatches.The additional bulge-inducing mismatches may be upstream and/ordownstream of the target adenosine. The bulges may be single-mismatchbulges (caused by one mismatching base pair) or multi-mismatch bulges(caused by more than one consecutive mismatching base pairs, preferablytwo or three consecutive mismatching base pairs).

The complementary RNA sequence in the dRNA is single-stranded. The dRNAmay be entirely single-stranded or have one or more (e.g., 1, 2, 3, ormore) double-stranded regions and/or one or more stem loop regions. Insome embodiments, the complementary RNA sequence is at least about anyone of 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200 or more nucleotides. In certainembodiments, the complementary RNA sequence is about any one of 40-260,45-250, 50-240, 60-230, 65-220, 70-220, 70-210, 70-200, 70-190, 70-180,70-170, 70-160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90,70-80, 75-200, 80-190, 85-180, 90-170, 95-160, 100-200, 100-150,100-175, 110-200, 110-175, 110-150, or 105-140 nucleotides in length. Insome embodiments, the dRNA is about 60-200 (such as about any of 60-150,65-140, 68-130, or 70-120) nucleotides long. In some embodiments, thecomplementary RNA sequence is about 71 nucleotides long. In someembodiments, the complementary RNA sequence is about 111 nucleotideslong.

In some embodiments, the dRNA, apart from the complementary RNAsequence, further comprises regions for stabilizing the dRNA, forexample, one or more double-stranded regions and/or stem loop regions.In some embodiments, the double-stranded region or stem loop region ofthe dRNA comprises no more than about any one of 200, 150, 100, 50, 40,30, 20, 10 or fewer base-pairs. In some embodiments, the dRNA does notcomprise a stem loop or double-stranded region. In some embodiments, thedRNA comprises an ADAR-recruiting domain. In some embodiments, the dRNAdoes not comprise an ADAR-recruiting domain.

The dRNA may comprise one or more modifications. In some embodiments,the dRNA has one or more modified nucleotides, including nucleobasemodification and/or backbone modification. In some embodiments, the dRNAis of about 60-200 nucleotides long and comprises one or moremoficiations (such as 2′-O-methylation and/or phosphorothioation). Insome embodiments, the modified dRNA comprises, from 5′ end to 3′ end: a5′ portion, a cytidine mismatch directly opposite the target A in thetarget RNA, and a 3′ portion, wherein the 3′ portion is no shorter thanabout 7 nt (such as no shorter than 8 nt, no shorter than 9 nt, and noshorter than 10 nt) nucleotides. In some embodiments, the 5′ portion isno shorter than about 25 (such as no shorter than about 30, no shorterthan about 35 nt, no shorter than about 40 nt, and no shorter than about45 nt) nucleotides. In some embodiments, the 5′ portion is about 25nt-85 nt nucleotides long (such as about 25 nt-80 nt, 25 nt-75 nt, 25nt-70 nt, 25 nt-65 nt, 25 nt-60 nt, 30 nt-55 nt, 40 nt-55 nt, or 45nt-55 nt nucleotides long). In some embodiments, the 3′ portion is about7 nt-25 nt nucleotide long (such as about 10 nt-15 nt or 21 nt-25 ntnucleotides long). In some embodiments, the 5′ portion is about 25 nt-85nt nucleotides long (such as about 25 nt-80 nt, 25 nt-75 nt, 25 nt-70nt, 25 nt-65 nt, 25 nt-60 nt, 30 nt-55 nt, 40 nt-55 nt, or 45 nt-55 ntnucleotides long), and the 3′ portion is about 7 nt-25 nt nucleotidelong (such as about 10 nt-15 nt or 21 nt-25 nt nucleotides long). Insome embodiments, the 5′ portion is longer than the 3′ portion. In someembodiments, the 5′ portion is about 55 nucleotides long, and the 3′portion is about 15 nucleotides long. In some embodiments, the positionof the cytidine mismatch in the dRNA is according to any of the dRNAsdescribed in the examples herein, and the dRNA can be, in the format ofXnt-c-Ynt, wherein X represents the length of the 5′ portion and Yrepresents the length of the 3′ portion: 55 nt-c-35 nt, 55 nt-c-25 nt,55 nt-c-24 nt, 55 nt-c-23 nt, 55 nt-c-22 nt, 55 nt-c-21 nt, 55 nt-c-20nt, 55 nt-c-19 nt, 55 nt-c-18 nt, 55 nt-c-17 nt, 55 nt-c-16 nt, 55nt-c-15 nt, 55 nt-c-14 nt, 55 nt-c-13 nt, 55 nt-c-12 nt, 55 nt-c-11 nt,55 nt-c-10 nt, 55 nt-c-9 nt, 55 nt-c-8 nt, 55 nt-c-7 nt, 55 nt-n-20 nt,50 nt-n-20 nt, 45 nt-n-20 nt, 55 nt-n-15 nt, 50 nt-n-15 nt, 45 nt-c-45nt, 45 nt-c-55 nt, 54 nt-c-12 nt, 53 nt-c-13 nt, 52 nt-c-14 nt, 51nt-c-15 nt, 50 nt-c-16 nt, 49 nt-c-17 nt, 48 nt-c-18 nt, 47 nt-c-19 nt,46 nt-c-20 nt, 45 nt-c-21 nt, 44 nt-c-22 nt, 43 nt-c-23 nt, 54 nt-c-15nt, 53 nt-c-16 nt, 52 nt-c-17 nt, 51 nt-c-18 nt, 50 nt-c-19 nt, 49nt-c-20 nt, 48 nt-c-21 nt, 47 nt-c-22 nt, 46 nt-c-23 nt, 54 nt-c-17 nt,53 nt-n-18 nt, 52 nt-n-19 nt, 51 nt-n-20 nt, 50 nt-n-21 nt, 49 nt-n-22nt, 48 nt-c-23.

In some embodiments, the dRNA is of about 60-200 nucleotides long andcomprises one or more moficiations (such as 2′-O-methylation and/orphosphorothioation). In some embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides and/orphosphorothiations in the first and last 3 internucleotide linkages. Insome embodiments, the dRNA comprises 2′-O-methylations in the first andlast 3 nucleotides, phosphorothiations in the first and last 3internucleotide linkages, and 2′-O-methylations in one or more uridines,for example on all uridines. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in a single or multiple or all uridines, and amodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine. In certain embodiments, the modification in thenucleotide opposite to the target adenosine, and/or one or twonucleotides most adjacent to the nucleotide opposite to the targetadenosine is a 2′-O-methylation. In certain embodiments, themodification in the nucleotide opposite to the target adenosine, and/orone or two nucleotides most adjacent to the nucleotide opposite to thetarget adenosine is a phosphorothiation linkage, such as a3′-phosphorothiation linkage. In certain embodiments, the dRNA comprises2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in all uridines, and a 2′-O-methylation in thenucleotide adjacent to the 3′ terminus or 5′ terminus of the nucleotideopposite to the target adenosine. In certain embodiments, the dRNAcomprises 2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in all uridines, and a 3′-phosphorothiation in thenucleotide opposite to the target adenosine and/or its 5′ and/or 3′ mostadjacent nucleotides. In some embodiments, the dRNA comprises2′-O-methylations in the first and last 5 nucleotides andphosphorothiations in the first and last 5 internucleotide linkages. Thepresent application also contemplates a construct comprising the dRNAdescribed herein. The term “construct” as used herein refers to DNA orRNA molecules that comprise a coding nucleotide sequence that can betranscribed into RNAs or expressed into proteins. In some embodiments,the construct contains one or more regulatory elements operably linkedto the nucleotide sequence encoding the RNA or protein. When theconstruct is introduced into a host cell, under suitable conditions, thecoding nucleotide sequence in the construct can be transcribed orexpressed.

In some embodiments, the construct comprises a promoter that is operablylinked, or spatially connected to the coding nucleotide sequence, suchthat the promoter controls the transcription or expression of the codingnucleotide sequence. A promoter may be positioned 5′ (upstream) of acoding nucleotide sequence under its control. The distance between thepromoter and the coding sequence may be approximately the same as thedistance between that promoter and the gene it controls in the gene fromwhich the promoter is derived. As is known in the art, variation in thisdistance may be accommodated without loss of promoter function. In someembodiments, the construct comprises a 5′ UTR and/or a 3′UTR thatregulates the transcription or expression of the coding nucleotidesequence.

In some embodiments, the construct is a vector encoding any one of thedRNAs disclosed in the present application. The term “vector” refers toa nucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g. circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the transcription or expression of coding nucleotide sequencesto which they are operatively linked. Such vectors are referred toherein as “expression vectors”.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for transcription or expression of thenucleic acid in a host cell. In some embodiments, the recombinantexpression vector includes one or more regulatory elements, which may beselected on the basis of the host cells to be used for transcription orexpression, which is operatively linked to the nucleic acid sequence tobe transcribed or expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor expression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell).

In some embodiments, there is provided a construct (e.g., vector, suchas viral vector) comprising a nucleotide sequence encoding the dRNA. Insome embodiments, there is provided a construct (e.g., vector, such asviral vector) comprising a nucleotide sequence encoding the ADAR. Insome embodiments, there is provided a construct comprising a firstnucleotide sequence encoding the dRNA and a second nucleotide sequenceencoding the ADAR. In some embodiments, the first nucleotide sequenceand the second nucleotide sequence are operably linked to the samepromoter. In some embodiments, the first nucleotide sequence and thesecond nucleotide sequence are operably linked to different promoters.In some embodiments, the promoter is inducible. In some embodiments, theconstruct does not encode for the ADAR. In some embodiments, the vectorfurther comprises nucleic acid sequence(s) encoding an inhibitor ofADAR3 (e.g., ADAR3 shRNA or siRNA) and/or a stimulator of interferon(e.g., IFN-α).

Methods of Treatment

The RNA editing methods and compositions described herein may be used totreat or prevent a disease or condition in an individual, including, butnot limited to hereditary genetic diseases and drug resistance.

In some embodiments, there is provided a method of editing a target RNAin a cell of an individual (e.g., human individual) ex vivo, comprisingediting the target RNA using any one of the methods of RNA editingdescribed herein.

In some embodiments, there is provided a method of editing a target RNAin a cell of an individual (e.g., human individual) ex vivo, comprisingintroducing a dRNA or a construct encoding the dRNA into the cell of theindividual, wherein the dRNA comprises a complementary RNA sequence thathybridizes to the target RNA, and wherein the dRNA is capable ofrecruiting an ADAR to deaminate a target A in the target RNA. In someembodiments, the target RNA is associated with a disease or condition ofthe individual. In some embodiments, the disease or condition is ahereditary genetic disease or a disease or condition associated with oneor more acquired genetic mutations (e.g., drug resistance). In someembodiments, the method further comprises obtaining the cell from theindividual.

In some embodiments, there is provided a method of treating orpreventing a disease or condition in an individual (e.g., humanindividual), comprising editing a target RNA associated with the diseaseor condition in a cell of the individual using any one of the methods ofRNA editing described herein.

In some embodiments, there is provided a method of treating orpreventing a disease or condition in an individual (e.g., humanindividual), comprising introducing a dRNA or a construct encoding thedRNA into an isolated cell of the individual ex vivo, wherein the dRNAcomprises a complementary RNA sequence that hybridizes to a target RNAassociated with the disease or condition, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA.In some embodiments, the ADAR is an endogenously expressed ADAR in theisolated cell. In some embodiments, the method comprises introducing theADAR or a construct encoding the ADAR to the isolated cell. In someembodiments, the method further comprises culturing the cell having theedited RNA. In some embodiments, the method further comprisesadministering the cell having the edited RNA to the individual. In someembodiments, the disease or condition is a hereditary genetic disease ora disease or condition associated with one or more acquired geneticmutations (e.g., drug resistance).

In some embodiments, there is provided a method of treating orpreventing a disease or condition in an individual (e.g., humanindividual), comprising introducing a dRNA or a construct encoding thedRNA into an isolated cell of the individual ex vivo, wherein the dRNAcomprises a complementary RNA sequence that hybridizes to a target RNAassociated with the disease or condition, and wherein the dRNA iscapable of recruiting an endogenously expressed ADAR of the host cell todeaminate a target A in the target RNA. In some embodiments, the methodfurther comprises culturing the cell having the edited RNA. In someembodiments, the method further comprises administering the cell havingthe edited RNA to the individual. In some embodiments, the disease orcondition is a hereditary genetic disease or a disease or conditionassociated with one or more acquired genetic mutations (e.g., drugresistance).

In some embodiments, there is provided a method of treating orpreventing a disease or condition in an individual (e.g., humanindividual), comprising administering an effective amount of a dRNA or aconstruct encoding the dRNA to the individual, wherein the dRNAcomprises a complementary RNA sequence that hybridizes to a target RNAassociated with the disease or condition, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA.In some embodiments, the ADAR is an endogenously expressed ADAR in thecells of the individual. In some embodiments, the method comprisesadministering the ADAR or a construct encoding the ADAR to theindividual. In some embodiments, the disease or condition is ahereditary genetic disease or a disease or condition associated with oneor more acquired genetic mutations (e.g., drug resistance).

Diseases and conditions suitable for treatment using the methods of thepresent application include diseases associated with a mutation, such asa G to A mutation, e.g., a G to A mutation that results in missensemutation, early stop codon, aberrant splicing, or alternative splicingin an RNA transcript. Examples of disease-associated mutations that maybe restored by the methods of the present application include, but arenot limited to, TP53^(W53X) (e.g., 158G>A) associated with cancer,IDUA^(W402X) (e.g., TGG>TAG Mutation in Exon 9) associated withMucopolysaccharidosis type I (MPS I), COL3A1^(1278X) (e.g., 3833G>Amutation) associated with Ehlers-Danlos syndrome, BMPR2^(W298X) (e.g.,893G>A) associated with primary pulmonary hypertension, AHI1^(W725X)(e.g., 2174G>A) associated with Joubert syndrome, FANCC^(W506X) (e.g.,1517G>A) associated with Fanconi anemia, MYBPC3^(W1098X) (e.g., 3293G>A)associated with primary familial hypertrophic cardiomyopathy, andIL2RG^(W237X) (e.g., 710G>A) associated with X-linked severe combinedimmunodeficiency. In some embodiments, the disease or condition is acancer. In some embodiments, the disease or condition is a monogeneticdisease. In some embodiments, the disease or condition is a polygeneticdisease.

In some embodiments, there is provided a method of treating a cancerassociated with a target RNA having a mutation (e.g., G>A mutation) inan individual, comprising introducing a dRNA or a construct encoding thedRNA into an isolated cell of the individual ex vivo, wherein the dRNAcomprises a complementary RNA sequence that hybridizes to the targetRNA, and wherein the dRNA is capable of recruiting an ADAR to deaminatea target A in the target RNA, thereby rescuing the mutation in thetarget RNA. In some embodiments, the ADAR is an endogenously expressedADAR in the isolated cell. In some embodiments, the method comprisesintroducing the ADAR or a construct encoding the ADAR to the isolatedcell. In some embodiments, the target RNA is TP53^(W53X) (e.g., 158G>A).In some embodiments, the dRNA comprises the nucleic acid sequence of SEQID NO: 195, 196 or 197.

In some embodiments, there is provided a method of treating orpreventing a cancer with a target RNA having a mutation (e.g., G>Amutation) in an individual, comprising administering an effective amountof a dRNA or a construct encoding the dRNA to the individual, whereinthe dRNA comprises a complementary RNA sequence that hybridizes to thetarget RNA associated with the disease or condition, and wherein thedRNA is capable of recruiting an ADAR to deaminate a target A in thetarget RNA, thereby rescuing the mutation in the target RNA. In someembodiments, the ADAR is an endogenously expressed ADAR in the cells ofthe individual. In some embodiments, the method comprises administeringthe ADAR or a construct encoding the ADAR to the individual. In someembodiments, the target RNA is TP53^(W53X) (e.g., 158G>A). In someembodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO:195, 196 or 197.

In some embodiments, there is provided a method of treating MPS I (e.g.,Hurler syndrome or Scheie syndrome) associated with a target RNA havinga mutation (e.g., G>A mutation) in an individual, comprising introducinga dRNA or a construct encoding the dRNA into an isolated cell of theindividual ex vivo, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA,thereby rescuing the mutation in the target RNA. In some embodiments,the ADAR is an endogenously expressed ADAR in the isolated cell. In someembodiments, the method comprises introducing the ADAR or a constructencoding the ADAR to the isolated cell. In some embodiments, the targetRNA is IDUA^(W402X) (e.g., TGG>TAG mutation in exon 9). In someembodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO:204 or 205.

In some embodiments, there is provided a method of treating orpreventing MPS I (e.g., Hurler syndrome or Scheie syndrome) with atarget RNA having a mutation (e.g., G>A mutation) in an individual,comprising administering an effective amount of a dRNA or a constructencoding the dRNA to the individual, wherein the dRNA comprises acomplementary RNA sequence that hybridizes to the target RNA associatedwith the disease or condition, and wherein the dRNA is capable ofrecruiting an ADAR to deaminate a target A in the target RNA, therebyrescuing the mutation in the target RNA. In some embodiments, the ADARis an endogenously expressed ADAR in the cells of the individual. Insome embodiments, the method comprises administering the ADAR or aconstruct encoding the ADAR to the individual. In some embodiments, thetarget RNA is IDUA^(W402X) (e.g., TGG>TAG mutation in exon 9). In someembodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO:204 or 205.

In some embodiments, there is provided a method of treating a disease orcondition Ehlers-Danlos syndrome associated with a target RNA having amutation (e.g., G>A mutation) in an individual, comprising introducing adRNA or a construct encoding the dRNA into an isolated cell of theindividual ex vivo, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA,thereby rescuing the mutation in the target RNA. In some embodiments,the ADAR is an endogenously expressed ADAR in the isolated cell. In someembodiments, the method comprises introducing the ADAR or a constructencoding the ADAR to the isolated cell. In some embodiments, the targetRNA is COL3A1^(W1278X) (e.g., 3833G>A mutation). In some embodiments,the dRNA comprises the nucleic acid sequence of SEQ ID NO: 198.

In some embodiments, there is provided a method of treating orpreventing Ehlers-Danlos syndrome with a target RNA having a mutation(e.g., G>A mutation) in an individual, comprising administering aneffective amount of a dRNA or a construct encoding the dRNA to theindividual, wherein the dRNA comprises a complementary RNA sequence thathybridizes to the target RNA associated with the disease or condition,and wherein the dRNA is capable of recruiting an ADAR to deaminate atarget A in the target RNA, thereby rescuing the mutation in the targetRNA. In some embodiments, the ADAR is an endogenously expressed ADAR inthe cells of the individual. In some embodiments, the method comprisesadministering the ADAR or a construct encoding the ADAR to theindividual. In some embodiments, the target RNA is COL3A1^(W1278X)(e.g., 3833G>A mutation). In some embodiments, the dRNA comprises thenucleic acid sequence of SEQ ID NO: 198.

In some embodiments, there is provided a method of treating primarypulmonary hypertension associated with a target RNA having a mutation(e.g., G>A mutation) in an individual, comprising introducing a dRNA ora construct encoding the dRNA into an isolated cell of the individual exvivo, wherein the dRNA comprises a complementary RNA sequence thathybridizes to the target RNA, and wherein the dRNA is capable ofrecruiting an ADAR to deaminate a target A in the target RNA, therebyrescuing the mutation in the target RNA. In some embodiments, the ADARis an endogenously expressed ADAR in the isolated cell. In someembodiments, the method comprises introducing the ADAR or a constructencoding the ADAR to the isolated cell. In some embodiments, the targetRNA is BMPR2^(W298X) (e.g., 893G>A). In some embodiments, the dRNAcomprises the nucleic acid sequence of SEQ ID NO: 199.

In some embodiments, there is provided a method of treating orpreventing primary pulmonary hypertension with a target RNA having amutation (e.g., G>A mutation) in an individual, comprising administeringan effective amount of a dRNA or a construct encoding the dRNA to theindividual, wherein the dRNA comprises a complementary RNA sequence thathybridizes to the target RNA associated with the disease or condition,and wherein the dRNA is capable of recruiting an ADAR to deaminate atarget A in the target RNA, thereby rescuing the mutation in the targetRNA. In some embodiments, the ADAR is an endogenously expressed ADAR inthe cells of the individual. In some embodiments, the method comprisesadministering the ADAR or a construct encoding the ADAR to theindividual. In some embodiments, the target RNA is BMPR2^(W298X) (e.g.,893G>A). In some embodiments, the dRNA comprises the nucleic acidsequence of SEQ ID NO: 199.

In some embodiments, there is provided a method of treating Joubertsyndrome associated with a target RNA having a mutation (e.g., G>Amutation) in an individual, comprising introducing a dRNA or a constructencoding the dRNA into an isolated cell of the individual ex vivo,wherein the dRNA comprises a complementary RNA sequence that hybridizesto the target RNA, and wherein the dRNA is capable of recruiting an ADARto deaminate a target A in the target RNA, thereby rescuing the mutationin the target RNA. In some embodiments, the ADAR is an endogenouslyexpressed ADAR in the isolated cell. In some embodiments, the methodcomprises introducing the ADAR or a construct encoding the ADAR to theisolated cell. In some embodiments, the target RNA is AHI1^(W725X)(e.g., 2174G>A). In some embodiments, the dRNA comprises the nucleicacid sequence of SEQ ID NO: 200.

In some embodiments, there is provided a method of treating orpreventing Joubert syndrome with a target RNA having a mutation (e.g.,G>A mutation) in an individual, comprising administering an effectiveamount of a dRNA or a construct encoding the dRNA to the individual,wherein the dRNA comprises a complementary RNA sequence that hybridizesto the target RNA associated with the disease or condition, and whereinthe dRNA is capable of recruiting an ADAR to deaminate a target A in thetarget RNA, thereby rescuing the mutation in the target RNA. In someembodiments, the ADAR is an endogenously expressed ADAR in the cells ofthe individual. In some embodiments, the method comprises administeringthe ADAR or a construct encoding the ADAR to the individual. In someembodiments, the target RNA is AHI1^(W725X) (e.g., 2174G>A). In someembodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO:200.

In some embodiments, there is provided a method of treating Fanconianemia associated with a target RNA having a mutation (e.g., G>Amutation) in an individual, comprising introducing a dRNA or a constructencoding the dRNA into an isolated cell of the individual ex vivo,wherein the dRNA comprises a complementary RNA sequence that hybridizesto the target RNA, and wherein the dRNA is capable of recruiting an ADARto deaminate a target A in the target RNA, thereby rescuing the mutationin the target RNA. In some embodiments, the ADAR is an endogenouslyexpressed ADAR in the isolated cell. In some embodiments, the methodcomprises introducing the ADAR or a construct encoding the ADAR to theisolated cell. In some embodiments, the target RNA is FANCC^(W506X)(e.g., 1517G>A). In some embodiments, the dRNA comprises the nucleicacid sequence of SEQ ID NO: 201.

In some embodiments, there is provided a method of treating orpreventing Fanconi anemia with a target RNA having a mutation (e.g., G>Amutation) in an individual, comprising administering an effective amountof a dRNA or a construct encoding the dRNA to the individual, whereinthe dRNA comprises a complementary RNA sequence that hybridizes to thetarget RNA associated with the disease or condition, and wherein thedRNA is capable of recruiting an ADAR to deaminate a target A in thetarget RNA, thereby rescuing the mutation in the target RNA. In someembodiments, the ADAR is an endogenously expressed ADAR in the cells ofthe individual. In some embodiments, the method comprises administeringthe ADAR or a construct encoding the ADAR to the individual. In someembodiments, the target RNA is FANCC^(W506X) (e.g., 1517G>A). In someembodiments, the dRNA comprises the nucleic acid sequence of SEQ ID NO:201.

In some embodiments, there is provided a method of treating primaryfamilial hypertrophic cardiomyopathy associated with a target RNA havinga mutation (e.g., G>A mutation) in an individual, comprising introducinga dRNA or a construct encoding the dRNA into an isolated cell of theindividual ex vivo, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA,thereby rescuing the mutation in the target RNA. In some embodiments,the ADAR is an endogenously expressed ADAR in the isolated cell. In someembodiments, the method comprises introducing the ADAR or a constructencoding the ADAR to the isolated cell. In some embodiments, the targetRNA is MYBPC3^(W1098X) (e.g., 3293G>A). In some embodiments, the dRNAcomprises the nucleic acid sequence of SEQ ID NO: 202.

In some embodiments, there is provided a method of treating orpreventing primary familial hypertrophic cardiomyopathy with a targetRNA having a mutation (e.g., G>A mutation) in an individual, comprisingadministering an effective amount of a dRNA or a construct encoding thedRNA to the individual, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA associated with the diseaseor condition, and wherein the dRNA is capable of recruiting an ADAR todeaminate a target A in the target RNA, thereby rescuing the mutation inthe target RNA. In some embodiments, the ADAR is an endogenouslyexpressed ADAR in the cells of the individual. In some embodiments, themethod comprises administering the ADAR or a construct encoding the ADARto the individual. In some embodiments, the target RNA isMYBPC3^(W1098X) (e.g., 3293G>A). In some embodiments, the dRNA comprisesthe nucleic acid sequence of SEQ ID NO: 202.

In some embodiments, there is provided a method of treating X-linkedsevere combined immunodeficiency associated with a target RNA having amutation (e.g., G>A mutation) in an individual, comprising introducing adRNA or a construct encoding the dRNA into an isolated cell of theindividual ex vivo, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA, and wherein the dRNA iscapable of recruiting an ADAR to deaminate a target A in the target RNA,thereby rescuing the mutation in the target RNA. In some embodiments,the ADAR is an endogenously expressed ADAR in the isolated cell. In someembodiments, the method comprises introducing the ADAR or a constructencoding the ADAR to the isolated cell. In some embodiments, the targetRNA is IL2RG^(W237X) (e.g., 710G>A). In some embodiments, the dRNAcomprises the nucleic acid sequence of SEQ ID NO: 203.

In some embodiments, there is provided a method of treating orpreventing X-linked severe combined immunodeficiency with a target RNAhaving a mutation (e.g., G>A mutation) in an individual, comprisingadministering an effective amount of a dRNA or a construct encoding thedRNA to the individual, wherein the dRNA comprises a complementary RNAsequence that hybridizes to the target RNA associated with the diseaseor condition, and wherein the dRNA is capable of recruiting an ADAR todeaminate a target A in the target RNA, thereby rescuing the mutation inthe target RNA. In some embodiments, the ADAR is an endogenouslyexpressed ADAR in the cells of the individual. In some embodiments, themethod comprises administering the ADAR or a construct encoding the ADARto the individual. In some embodiments, the target RNA is IL2RG^(W237X)(e.g., 710G>A). In some embodiments, the dRNA comprises the nucleic acidsequence of SEQ ID NO: 203.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing one moresymptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the occurrence orrecurrence of the disease, delay or slowing the progression of thedisease, ameliorating the disease state, providing a remission (whetherpartial or total) of the disease, decreasing the dose of one or moreother medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. Also encompassed by “treatment” is a reduction ofpathological consequence of the disease or condition. The methods of theinvention contemplate any one or more of these aspects of treatment.

The terms “individual,” “subject” and “patient” are used interchangeablyherein to describe a mammal, including humans. An individual includes,but is not limited to, human, bovine, horse, feline, canine, rodent, orprimate. In some embodiments, the individual is human. In someembodiments, an individual suffers from a disease or condition, such asdrug resistance. In some embodiments, the individual is in need oftreatment.

As is understood in the art, an “effective amount” refers to an amountof a composition (e.g., dRNA or constructs encoding the dRNA) sufficientto produce a desired therapeutic outcome (e.g., reducing the severity orduration of, stabilizing the severity of, or eliminating one or moresymptoms of a disease or condition). For therapeutic use, beneficial ordesired results include, e.g., decreasing one or more symptoms resultingfrom the disease (biochemical, histologic and/or behavioral), includingits complications and intermediate pathological phenotypes presentedduring development of the disease, increasing the quality of life ofthose suffering from the disease or condition, decreasing the dose ofother medications required to treat the disease, enhancing effect ofanother medication, delaying the progression of the disease, and/orprolonging survival of patients.

Generally, dosages, schedules, and routes of administration of thecompositions (e.g., dRNA or construct encoding dRNA) may be determinedaccording to the size and condition of the individual, and according tostandard pharmaceutical practice. Exemplary routes of administrationinclude intravenous, intra-arterial, intraperitoneal, intrapulmonary,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal.

The RNA editing methods of the present application can not only be usedin animal cells, for example mammalian cells, but also may be used inmodification of RNAs of plant or fungi, for example, in plants or fungithat have endogenously expressed ADARs. The methods described herein canbe used to generate genetically engineered plant and fungi with improvedproperties.

Compositions, Kits and Articles of Manufacture

Also provided herein are compositions (such as pharmaceuticalcompositions) comprising any one of the dRNAs, constructs, libraries, orhost cells having edited RNA as described herein.

In some embodiments, there is provided a pharmaceutical compositioncomprising any one of the dRNAs or constructs encoding the dRNAdescribed herein, and a pharmaceutically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propylparaben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such asolyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some embodiments,lyophilized formulations are provided. Pharmaceutical compositions to beused for in vivo administration must be sterile. This is readilyaccomplished by, e.g., filtration through sterile filtration membranes.

Further provided are kits useful for any one of the methods of RNAediting or methods of treatment described herein, comprising any one ofthe dRNAs, constructs, compositions, libraries, or edited host cells asdescribed herein.

In some embodiments, there is provided a kit for editing a target RNA ina host cell, comprising a dRNA, wherein the dRNA comprises acomplementary RNA sequence that hybridizes to the target RNA, whereinthe dRNA is capable of recruiting an ADAR to deaminate an A in thetarget RNA. In some embodiments, the kit further comprises an ADAR or aconstruct encoding an ADAR. In some embodiments, the kit furthercomprises an inhibitor of ADAR3 or a construct thereof. In someembodiments, the kit further comprises a stimulator of interferon or aconstruct thereof. In some embodiments, the kit further comprises aninstruction for carrying out any one of the RNA editing methodsdescribed herein.

The kits of the present application are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Kits may optionally provide additional components such as transfectionor transduction reagents, cell culturing medium, buffers, andinterpretative information.

The present application thus also provides articles of manufacture. Thearticle of manufacture can comprise a container and a label or packageinsert on or associated with the container. Suitable containers includevials (such as sealed vials), bottles, jars, flexible packaging, and thelike. In some embodiments, the container holds a pharmaceuticalcomposition, and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). The container holding thepharmaceutical composition may be a multi-use vial, which allows forrepeat administrations (e.g. from 2-6 administrations) of thereconstituted formulation. Package insert refers to instructionscustomarily included in commercial packages of therapeutic products thatcontain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch products. Additionally, the article of manufacture may furthercomprise a second container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes.

The kits or article of manufacture may include multiple unit doses ofthe pharmaceutical compositions and instructions for use, packaged inquantities sufficient for storage and use in pharmacies, for example,hospital pharmacies and compounding pharmacies.

EXEMPLARY EMBODIMENTS

Among the embodiments provided herein are:

-   -   1. A method for editing a target RNA in a host cell, comprising        introducing a deaminase-recruiting RNA (dRNA) or a construct        encoding the dRNA into the host cell, wherein the dRNA comprises        a complementary RNA sequence that hybridizes to the target RNA,        and wherein the deaminase-recruiting RNA is capable of        recruiting an adenosine deaminase acting on RNA (ADAR) to        deaminate a target adenosine in the target RNA.    -   2. The method of embodiment 1, wherein the RNA sequence        comprises a cytidine, adenosine or uridine directly opposite the        target adenosine in the target RNA.    -   3. The method of embodiment 2, wherein the RNA sequence        comprises a cytidine mismatch directly opposite the target        adenosine in the target RNA.    -   4. The method of embodiment 3, wherein the cytidine mismatch is        located at least 20 nucleotides away from the 3′ end of the        complementary sequence, and at least 5 nucleotides away from the        5′ end of the complementary sequence in the dRNA.    -   5. The method of embodiment 4, wherein the cytidine mismatch is        located within 10 nucleotides from the center (e.g., at the        center) of the complementary sequence in the dRNA.    -   6. The method of any one of embodiments 1-5, wherein the RNA        sequence further comprises one or more guanosines each opposite        a non-target adenosine in the target RNA.    -   7. The method of any one of embodiments 1-6, wherein the        complementary sequence comprises two or more consecutive        mismatch nucleotides opposite a non-target adenosine in the        target RNA.    -   8. The method of any one of embodiments 1-7, wherein the 5′        nearest neighbor of the target adenosine in the target RNA is a        nucleotide selected from U, C, A and G with the preference        U>C≈A>G and the 3′ nearest neighbor of the target adenosine in        the target RNA is a nucleotide selected from Q C, A and U with        the preference G>C>A≈U.    -   9. The method of any one of embodiments 1-8, wherein the target        adenosine is in a three-base motif selected from the group        consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG AAC,        AAA, AAU, GAG, GAC, GAA and GAU in the target RNA.    -   10. The method of embodiment 9, wherein the three-base motif is        UAQ and wherein the deaminase-recruiting RNA comprises an A        directly opposite the uridine in the three-base motif, a        cytidine directly opposite the target adenosine, and a cytidine,        guanosine or uridine directly opposite the guanosine in the        three-base motif.    -   11. The method of any one of embodiments 1-10, wherein the        deaminase-recruiting RNA is about 40-260 nucleotides in length.    -   12. The method of embodiment 11, wherein the        deaminase-recruiting RNA is about 60-230 nucleotides in length.    -   13. The method of embodiment 11 or 12, wherein the dRNA is more        than about 60 nucleotides in length.    -   14. The method of any one of embodiments 11-13, wherein the dRNA        is about 100 to about 150 (e.g., about 110-150) nucleotides in        length.    -   15. The method of any one of embodiments 1-14, wherein the        target RNA is an RNA selected from the group consisting of a        pre-messenger RNA, a messenger RNA, a ribosomal RNA, a transfer        RNA, a long non-coding RNA and a small RNA.    -   16. The method of embodiment 15, wherein the target RNA is a        pre-messenger RNA.    -   17. The method of any one of embodiments 1-16, wherein the ADAR        is endogenously expressed by the host cell.    -   18. The method of any one of embodiments 1-16, wherein the ADAR        is exogenous to the host cell.    -   19. The method of embodiment 18, further comprising introducing        the ADAR to the host cell.    -   20. The method of embodiment 18 or 19, wherein the ADAR        comprises an E1008 mutation.    -   21. The method of any one of embodiments 1-20, wherein the        deaminase-recruiting RNA is a single-stranded RNA.    -   22. The method of any one of embodiments 1-20, wherein the        complementary RNA sequence is single-stranded, and wherein the        deaminase-recruiting RNA further comprises one or more        double-stranded regions.    -   23. The method of any one of embodiments 1-22, wherein the dRNA        does not comprise an ADAR-recruiting domain (e.g., a DSB-binding        domain, a GluR2 domain, or a MS2 domain).    -   24. The method of any one of embodiments 1-23, wherein the dRNA        does not comprise a chemically modified nucleotide (e.g.,        2′-O-methylation or phosphorothioation).    -   25. The method of embodiment 26, wherein the deamination of the        target adenosine in the target RNA results in point mutation,        truncation, elongation and/or misfolding of the protein encoded        by the target RNA, or a functional, full-length,        correctly-folded and/or wild-type protein by reversal of a        missense mutation, an early stop codon, aberrant splicing, or        alternative splicing in the target RNA.    -   26. The method of any one of embodiments 1-27, wherein the host        cell is a eukaryotic cell.    -   27. The method of embodiment 28, wherein the host cell is a        mammalian cell.    -   28. The method of embodiment 29, wherein the host cell is a        human or mouse cell.    -   29. The method of embodiment 29 or 30, wherein the ADAR is ADAR1        and/or ADAR2.    -   30. The method of any one of embodiments 1-31, wherein the host        cell is a primary cell.    -   31. The method of embodiment 32, wherein the host cell is a T        cell.    -   32. The method of embodiment 32, wherein the host cell is a        post-mitotic cell.    -   33. The method of any one of embodiments 1-34, further        comprising introducing an inhibitor of ADAR3 to the host cell.    -   34. The method of any one of embodiments 1-35, further        comprising introducing a stimulator of interferon to the host        cell.    -   35. The method of any one of embodiments 1-36, comprising        introducing a plurality of dRNAs each targeting a different        target RNA.    -   36. The method of any one of embodiments 1-37, wherein the        efficiency of editing the target RNA is at least about 30%        (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,        70%, 75%, 80%, 85%, 90% or higher).    -   37. The method of any one of embodiments 1-38, wherein the dRNA        does not induce immune response.    -   38. An edited RNA or a host cell having an edited RNA produced        by the method of any one of embodiments 1-39.    -   39. A method for treating or preventing a disease or condition        in an individual, comprising editing a target RNA associated        with the disease or condition in a cell of the individual        according to any one of the embodiments 1-39.    -   40. The method of embodiment 41, wherein the disease or        condition is a hereditary genetic disease or a disease or        condition associated with one or more acquired genetic        mutations.    -   41. The method of embodiment 41 or 42, wherein the target RNA        has a G to A mutation.    -   42. The method of any one of embodiments 41-43, wherein disease        or condition is a monogenetic disease or condition.    -   43. The method of any one of embodiments 41-44, wherein the        disease or condition is a polygenetic disease or condition.    -   44. The method of any one of embodiments 41-45, wherein:    -   (i) the target RNA is TP53, and the disease or condition is        cancer;    -   (ii) the target RNA is IDUA, and the disease or condition is        Mucopolysaccharidosis type I (MPS I);    -   (iii) the target RNA is COL3A1, and the disease or condition is        Ehlers-Danlos syndrome;    -   (iv) the target RNA is BMPR2, and the disease or condition is        Joubert syndrome;    -   (v) the target RNA is FANCC, and the disease or condition is        Fanconi anemia;    -   (vi) the target RNA is MYBPC3, and the disease or condition is        primary familial hypertrophic cardiomyopathy; or    -   (vii) the target RNA is IL2RG, and the disease or condition is        X-linked severe combined immunodeficiency.    -   45. A deaminase-recruiting RNA (dRNA) for deamination of a        target adenosine in a target RNA by recruiting an Adenosine        Deaminase Acting on RNA (ADAR), comprising a complementary RNA        sequence that hybridizes to the target RNA.    -   46. The deaminase-recruiting RNA of embodiment 47, wherein the        RNA sequence comprises a cytosine, adenosine or U directly        opposite the target adenosine in the target RNA.    -   47. The dRNA of embodiment 48, wherein the RNA sequence        comprises a cytidine mismatch directly opposite the target        adenosine in the target RNA.    -   48. The dRNA of embodiment 49, wherein the cytidine mismatch is        located at least 20 nucleotides away from the 3′ end of the        complementary sequence, and at least 5 nucleotides away from the        5′ end of the complementary sequence in the dRNA.    -   49. The dRNA of embodiment 50, wherein the cytidine mismatch is        located within 10 nucleotides from the center (e.g., at the        center) of the complementary sequence in the dRNA.    -   50. The deaminase-recruiting RNA of any one of embodiments        47-51, wherein the RNA sequence further comprises one or more        guanosines each directly opposite a non-target adenosine in the        target RNA.    -   51. The dRNA of any one of embodiments 47-51, wherein the        complementary sequence comprises two or more consecutive        mismatch nucleotides opposite a non-target adenosine in the        target RNA.    -   52. The deaminase-recruiting RNA of any one of embodiments        47-53, wherein the target adenosine is in a three-base motif        selected from the group consisting of UAQ UAC, UAA, UAU, CAG,        CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU in the        target RNA.    -   53. The deaminase-recruiting RNA of embodiment 54, wherein the        three-base motif is UAQ and wherein the dRNA comprises an        adenosine directly opposite the uridine in the three-base motif,        a cytosine directly opposite the target adenosine, and a        cytidine, guanosine or uridine directly opposite the guanosine        in the three-base motif.    -   54. The deaminase-recruiting RNA of embodiment 55, wherein the        three-base motif is UAG in the target RNA, and wherein the        deaminase-recruiting RNA comprises ACC, ACG or ACU opposite the        UAG of the target RNA.    -   55. The deaminase-recruiting RNA of any one of embodiments        47-56, wherein the deaminase-recruiting RNA is about 40-260        nucleotides in length.    -   56. The dRNA of embodiment 57, wherein the dRNA is more than        about 70 nucleotides in length.    -   57. The dRNA of embodiment 57 or 58, wherein the dRNA is about        100 to about 150 nucleotides (e.g., about 110-150) in length.    -   58. The dRNA of any one of embodiments 47-59, wherein the dRNA        does not comprise an ADAR-recruiting domain (e.g., a DSB-binding        domain, a GluR2 domain, or a MS2 domain).    -   59. The dRNA of any one of embodiments 47-60, wherein the dRNA        does not comprise a chemically modified nucleotide (e.g.,        2′-O-methylation or phosphorothioation).    -   60. A construct encoding the deaminase-recruiting RNA of any one        of embodiments 47-61.    -   61. The construct of embodiment 62, wherein the construct is a        viral vector (e.g., lentiviral vector) or a plasmid.    -   62. A library comprising a plurality of the deaminase-recruiting        RNAs of any one of embodiments 47-61 or the construct of        embodiment 62 or 63.    -   63. A composition comprising the deaminase-recruiting RNA of any        one of embodiments 47-61, the construct of embodiment 62 or 63,        or the library of embodiment 64.    -   64. A host cell comprising the deaminase-recruiting RNA of any        one of embodiments 47-61 or the construct of embodiment 62 or        63.    -   65. The host cell of embodiment 66, wherein the host cell is a        eukaryotic cell.    -   66. The host cell of embodiment 66 or 67, wherein the host cell        is a primary cell.    -   67. A kit for editing a target RNA in a host cell, comprising a        deaminase-recruiting RNA, wherein the deaminase-recruiting RNA        comprises a complementary RNA sequence that hybridizes to the        target RNA, wherein the deaminase-recruiting RNA is capable of        recruiting an ADAR to deaminate a target adenosine in the target        RNA.    -   68. A deaminase-recruiting RNA (dRNA) of 60-200 nucleotides,        wherein:    -   1) the dRNA comprises a complementary RNA sequence capable of        hybridizing to a target RNA;    -   2) the dRNA is capable of recruiting a deaminase or a construct        comprising a deaminase or a construct comprising a catalytic        domain of a deaminase to deaminate a target adenosine in the        target RNA;    -   3) the dRNA comprises one or more chemical modifications.    -   69. The dRNA of embodiment 68, wherein the dRNA is longer than        about any of 60 nt, 65 nt, 70 nt, 80 nt, 90 nt, 100 nt, or 110        nt.    -   70. The dRNA of embodiment 1 or embodiment 69, comprising one or        more mismatches, wobbles and/or bulges with the complementary        target RNA region.    -   71. The dRNA of any one of embodiments 68-70, wherein the        complementary RNA sequence comprises a cytidine, adenosine or        uridine directly opposite to a target adenosine in the target        RNA.    -   72. The dRNA of embodiment 71, wherein the cytidine, adenosine        or uridine directly opposite to the target adenosine locates at        least about 7 nucleotides away from the 3′ end, for example at        least about 8, 9, 10 or more nucleotides from the 3′ end, or        about 7-25 nt from the 3′ end.    -   73. The dRNA of any one of embodiments 71-72, wherein the        cytidine, adenosine or uridine directly opposite to the target        adenosine locates at least about 25 nucleotides away from the 5′        end, for example at least about 30, 35, 40, 45, 50 or 55        nucleotides from the 5′ end, or about 45-55 nt from the 5′ end.    -   74. The dRNA of any of embodiments 71-73, wherein the lengths of        the 5′ and 3′ sequences flanking the cytidine, adenosine or        uridine directly opposite to the target adenosine are unequal.    -   75. The dRNA of any of embodiments 71-74, wherein the length of        the 5′ sequence flanking the cytidine, adenosine or uridine        directly opposite to the target adenosine is longer than the 3′        sequence.    -   76. The dRNA of any one of embodiments 68-75, comprising a        cytidine directly opposite to the target adenosine in the target        RNA.    -   77. The dRNA of any one of embodiments 68-76, wherein the        complementary RNA sequence comprises one or more guanosines each        opposite to a non-target adenosine in the target RNA.    -   78. The dRNA of any one of embodiments 68-77, wherein the        complementary sequence comprises two or more consecutive        mismatch nucleotides opposite to a non-target adenosine in the        target RNA.    -   79. The dRNA of any one of embodiments 68-78, wherein the 5′        nearest neighbor of the target adenosine in the target RNA is a        nucleotide selected from U, C, A and G with the preference        U>C≈A>G and the 3′ nearest neighbor of the target adenosine in        the target RNA is a nucleotide selected from Q C, A and U with        the preference G>C>A≈U.    -   80. The dRNA of any one of embodiments 68-79, wherein the target        adenosine is in a three-base motif selected from the group        consisting of UAQ UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC,        AAA, AAU, GAG, GAC, GAA and GAU in the target RNA.    -   81. The dRNA of embodiment 80, wherein the three-base motif is        UAQ and wherein the dRNA comprises an A directly opposite to the        uridine in the three-base motif, a cytidine directly opposite to        the target adenosine, and a cytidine, guanosine or uridine        directly opposite the guanosine in the three-base motif.    -   82. The dRNA of embodiment 81, comprising a 5′-CCA-3′ directly        opposite to the three-base motif of UAG.    -   83. The dRNA of any one of embodiments 68-82, wherein the        chemical modification is methylation and/or phosphorothioation,        for example 2′-O-methylation and/or internucleotide        phosphorothioate linkage.    -   84. The dRNA of embodiment 83, wherein the chemical modification        comprises a 2′-O-methylation in the first and last 1-5, 2-5,        3-5, 4-5 nucleotides and/or phosphorothioations in the first and        last 1-5, 2-5, 3-5, 4-5 internucleotide linkages.    -   85. The dRNA of embodiment 83 or embodiment 84, wherein the        chemical modification comprises a 2′-O-methylation and/or a        3′-phosphorothioation in the nucleotide opposite to the target        adenosine and/or its 5′ and/or 3′ most adjacent nucleotides.    -   86. The dRNA of any one of embodiments 1-85, the chemical        modification is selected from a group consisting of:    -   1) 2′-G-methylations in the first and last 3 nucleotides and/or        phosphorothiations in the first and last 3 internucleotide        linkages;    -   2) 2′-O-methylations in the first and last 3 nucleotides,        phosphorothiations in the first and last 3 internucleotide        linkages, and 2′-O-methylations in one or more uridines, for        example on all uridines;    -   3) 2′-O-methylations in the first and last 3 nucleotides,        phosphorothiations in the first and last 3 internucleotide        linkages, 2′-O-methylations in a single or multiple or all        uridines, and a modification in the nucleotide opposite to the        target adenosine, and/or its 5′ and/or 3′ most adjacent        nucleotides;    -   4) 2′-O-methylations in the first and last 3 nucleotides,        phosphorothiations in the first and last 3 internucleotide        linkages, 2′-O-methylations in all uridines, and a        2′-O-methylation in the nucleotide most adjacent to the 3′        terminus and/or 5′ terminus of the nucleotide opposite to the        target adenosine;    -   5) 2′-O-methylations in the first and last 3 nucleotides,        phosphorothiations in the first and last 3 internucleotide        linkages, 2′-O-methylations in all uridines, and a 3′        phosphorothiation in the nucleotide opposite to the target        adenosine and/or its 5′ and/or 3′ most adjacent nucleotides; and    -   6) 2′-O-methylations in the first and last 5 nucleotides and        phosphorothiations in the first and last 5 internucleotide        linkages.    -   87. The dRNA of embodiment 86, wherein the modification in the        nucleotide opposite to the target adenosine, and/or one or two        nucleotides most adjacent to the nucleotide opposite to the        target adenosine is 2′-O-methylation or phosphorothiation        linkage, such as a 3′-phosphorothiation linkage.    -   88. The dRNA of any one of embodiments 68-87, which does not        comprise an ADAR-recruiting domain capable of forming an        intramolecular stem loop structure for binding an ADAR enzyme.    -   89. A construct comprising or encoding a dRNA of any one of        claims 68-88.    -   90. A method for editing a target RNA in a host cell, comprising        introducing a dRNA of any one of embodiments 68-89 into host        cells, including, but not limited to eukaryotic cell, primary        cell, T cell, mammalian cell, human cell, murine cell, etc., by        infection, electrotransfection, lipofection, endocytosis,        liposome or lipid nanoparticle delivery, etc.    -   91. The method of embodiment 90, further comprises introducing        an inhibitor of ADAR3 to the host cell.    -   92. The method of embodiment 90 or embodiment 91, further        comprises introducing a stimulator of interferon to the host        cell.    -   93. The method of any one of embodiments 90-92, comprising        introducing a plurality of the dRNAs each targeting a different        target RNA.    -   94. The method of any one of embodiments 90-93, wherein the dRNA        does not induce immune response.    -   95. The method of any one of embodiments 90-94, further        comprises introducing an exogenous ADAR to the host cell.    -   96. The method of embodiment 95, wherein the ADAR is an ADAR1        comprising an E1008 mutation.    -   97. A composition, cell, library or kit comprising the dRNAs of        any one of embodiments 68-89

EXAMPLE

The examples below are intended to be purely exemplary of the presentapplication and should therefore not be considered to limit theinvention in any way. The following examples and detailed descriptionare offered by way of illustration and not by way of limitation.

Materials and Methods Plasmids Construction

The dual fluorescence reporter was cloned by PCR amplifying mCherry andEGFP (the EGFP first codon ATG was deleted) coding DNA, the 3×GS linkerand targeting DNA sequence were added via primers during PCR. Then thePCR products were cleaved and linked by Type IIs restriction enzymeBsmB1 (Thermo) and T4 DNA ligase (NEB), which then were inserted intopLenti backbone (pLenti-CMV-MCS-SV-Bsd, Stanley Cohen Lab, StanfordUniversity).

The dLbuCas13 DNA was PCR amplified from the Lbu plasmids (Addgene#83485). The ADAR1DD and ADAR2DD were amplified from ADAR1(p150) cDNAand ADAR2 cDNA, both of which were gifts from Han's lab at XiamenUniversity. The ADAR1DD or ADAR2DD were fused to dLbuCas13 DNA byoverlap-PCR, and the fused PCR products were inserted into pLentibackbone.

For expression of dRNA in mammalian cells, the dRNA sequences weredirectly synthesized (for short dRNAs) and annealed or PCR amplified bysynthesizing overlapping ssDNA, and the products were cloned into thecorresponding vectors under U6 expression by Golden-gate cloning.

The full length ADAR1(p110) and ADAR1(p150) were PCR amplified fromADAR1(p150) cDNA, and the full length ADAR2 were PCR amplified fromADAR2 cDNA, which were then cloned into pLenti backbone, respectively.

For the three versions of dual fluorescence reporters (Reporter-1, -2and -3), mCherry and EGFP (the start codon ATG of EGFP was deleted)coding sequences were PCR amplified, digested using BsmBI (Thermo FisherScientific, ER0452), followed by T4 DNA ligase (NEB, M0202L)-mediatedligation with GGGGS linkers. The ligation product was subsequentlyinserted into the pLenti-CMV-MCS-PURO backbone.

For the dLbuCas13-ADAR_(DD) (E1008Q) expressing construct, theADAR1_(DD) gene was amplified from the ADAR1^(p150) construct (a giftfrom Jiahuai Han's lab, Xiamen University). The dLbuCas13 gene wasamplified by PCR from the Lbu_C2c2_R472A_H477A_R1048A_H1053A plasmid(Addgene #83485). The ADAR1_(DD) (hyperactive E1008Q variant) wasgenerated by overlap-PCR and then fused to dLbuCas13. The ligationproducts were inserted into the pLenti-CMV-MCS-BSD backbone.

For arRNA-expressing construct, the sequences of arRNAs were synthesizedand golden-gate cloned into the pLenti-sgRNA-lib 2.0 (Addgene #89638)backbone, and the transcription of arRNA was driven by hU6 promoter. Forthe ADAR expressing constructs, the full length ADAR1^(p110) andADAR1^(p150) were PCR amplified from the ADAR1^(p150) construct, and thefull length ADAR2 were PCR amplified from the ADAR2 construct (a giftfrom Jiahuai Han's lab, Xiamen University). The amplified products werethen cloned into the pLenti-CMV-MCS-BSD backbone.

For the constructs expressing genes with pathogenic mutations, fulllength coding sequences of TP53 (ordered from Vigenebio) and other 6disease-relevant genes (COL3A1, BMPR2, AHI1, FANCC, MYBPC3 and IL2RGgifts from Jianwei Wang's lab, Institute of pathogen biology, ChineseAcademy of Medical Sciences) were amplified from the constructs encodingthe corresponding genes with introduction of G>A mutations throughmutagenesis PCR. The amplified products were cloned into thepLenti-CMV-MCS-mCherry backbone through Gibson cloning method⁵⁹.

Mammalian Cell Lines and Cell Culture

Mammalian cell lines were cultured Dulbecco's Modified Eagle Medium(10-013-CV, Corning, Tewksbury, Mass., USA), adding 10% fetal bovineserum (Lanzhou Bailing Biotechnology Co., Ltd., Lanzhou, China),supplemented with 1% penicillin-streptomycin under 5% CO₂ at 37° C. TheADAR1-KO cell line was purchased from EdiGene China, and the genotypingresults were also provided by EdiGene China.

The HeLa and B16 cell lines were from Z. Jiang's laboratory (PekingUniversity). And the HEK293T cell line was from C. Zhang's laboratory(Peking University). RD cell line was from J Wang's laboratory(Institute of Pathogen Biology, Peking Union Medical College & ChineseAcademy of Medical Sciences). SF268 cell lines were from Cell Center,Institute of Basic Medical Sciences, Chinese Academy of MedicalSciences. A549 and SW13 cell lines were from EdiGene Inc. HepG2, HT29,NIH3T3, and MEF cell lines were maintained in our laboratory at PekingUniversity. These mammalian cell lines were cultured in Dulbecco'sModified Eagle Medium (Corning, 10-013-CV) with 10% fetal bovine serum(CellMax, SA201.02), additionally supplemented with 1%penicillin-streptomycin under 5% CO₂ at 37° C. Unless otherwisedescribed, cells were transfected with the X-tremeGENE HP DNAtransfection reagent (Roche, 06366546001) according to themanufacturer's instruction.

The human primary pulmonary fibroblasts (#3300) and human primarybronchial epithelial cells (#3210) were purchased from ScienCellResearch Laboratories, Inc. and were cultured in Fibroblast Medium(ScienCell, #2301) and Bronchial Epithelial Cell Medium (ScienCell,#3211), respectively. Both media were supplemented with 15% fetal bovineserum (BI) and 1% penicillin-streptomycin. The primary GM06214 (Hurlersyndrome patient derived fibroblast; homozygous of a TGG>TAG mutation atnucleotide 1293 in exon 9 of the IDUA gene [Trp402Ter (W402X)]) andGM01323 (Scheie syndrome patient derived fibroblast, having 0.3% IDUAactivity compared to WT cells. Much milder symptoms than Hurlersyndrome. Compound heterozygote: a G>A transition in intron 5, inposition −7 from exon 6 (IVS5AS-7G>A) and TGG>TAG at nucleotide 1293 inexon 9 of the IDUA gene [Trp402Ter (W402X)]. Serving as a positivecontrol in examples in this invention) cells were ordered from CoriellInstitute for Medical Research and cultured in Dulbecco's Modified EagleMedium (Corning, 10-013-CV) with 15% fetal bovine serum (BI) and 1%penicillin-streptomycin. All cells were cultured under 5% CO₂ at 37° C.

Reporter System Transfection, FACS Analysis and Sanger Sequencing

For dual fluorescence reporter editing experiments, 293T-WT cells or293T-ADAR1-KO cells were seeded in 6 wells plates (6×10⁵ cells/well), 24hours later, 1.5 μg reporter plasmids and 1.5 μg dRNA plasmids wereco-transfected using the X-tremeGENE HP DNA transfection reagent(06366546001; Roche, Mannheim, German), according to the supplier'sprotocols. 48 to 72 hours later, collected cells and performed FACSanalysis. For further confirming the reporter mRNA editing, we sortedthe EGFP-positive cells from 293T-WT cells transfected with reporter anddRNA plasmids using a FACS Aria flow cytometer (BD Biosciences),followed by total RNA isolation (TIANGEN, DP430). Then the RNA wasreverse-transcribed into cDNA via RT-PCR (TIANGEN, KR103-04), and thetargeted locus were PCR amplified with the corresponding primer pairs(23 PCR cycles) and the PCR products were purified for Sangersequencing.

For ADAR1(p110), ADAR1(p150) or ADAR2 rescue and overexpressionexperiments, 293T-WT cells or 293T-ADAR1-KO cells were seeded in 12wells plates (2.5×10⁵ cells/well), 24 hours later, 0.5 μg reporterplasmids, 0.5 μg dRNA plasmids and 0.5 μg ADAR1/2 plasmids (pLentibackbone as control) were co-transfected using the X-tremeGENE HP DNAtransfection reagent (06366546001, Roche, Mannheim, German). 48 to 72hours later, collected cells and performed FACS analysis.

For endogenous mRNA experiments, 293T-WT cells were seeded in 6 wellsplates (6×10⁵ cells/well), When approximately 70% confluent, 3 μg dRNAplasmids were transfected using the X-tremeGENE HP DNA transfectionreagent (06366546001, Roche, Mannheim, German). 72 hours later,collected cells and sorted GFP-positive or BFP-positive cells (accordingto the corresponding fluorescence maker) via FACS for the following RNAisolation.

Isolation and Culture of Human Primary T Cells

Primary human T cells were isolated from leukapheresis products fromhealthy human donor. Briefly, Peripheral blood mononuclear cells (PBMCs)were isolated by Ficoll centrifugation (Dakewei, AS1114546), and T cellswere isolated by magnetic negative selection using an EasySep Human TCell Isolation Kit (STEMCELL, 17951) from PBMCs. After isolation, Tcells were cultured in X-vivo15 medium, 10% FBS and IL2 (1000 U/ml) andstimulated with CD3/CD28 DynaBeads (ThermoFisher, 11131D) for 2 days.Leukapheresis products from healthy donors were acquired from AllCellsLLC China. All healthy donors provided informed consent.

Lenti-Virus Package and Reporter Cells Line Construction

The expression plasmid was co-transfected into HEK293T-WT cells,together with two viral packaging plasmids, pR8.74 and pVSVG (Addgene)via the X-tremeGENE HP DNA transfection reagent. 72 hours later, thesupernatant virus was collected and stored at −80° C. The HEK293T-WTcells were infected with lenti-virus, 72 hours later, mCherry-positivecells were sorted via FACS and cultured to select a single clone celllines stably expressing dual fluorescence reporter system with much lowEGFP background by limiting dilution method.

For the stable reporter cell lines, the reporter constructs(pLenti-CMV-MCS-PURO backbone) were co-transfected into HEK293T cells,together with two viral packaging plasmids, pR8.74 and pVSVG. 72 hourslater, the supernatant virus was collected and stored at −80° C. TheHEK293T cells were infected with lentivirus, then mCherry-positive cellswere sorted via FACS and cultured to select a single clone cell linesstably expressing dual fluorescence reporter system without detectableEGFP background. The HEK293T ADAR-1^(−/−) and TP53^(−/−) cell lines weregenerated according to a previously reported method⁶⁰. ADAR1-targetingsgRNA and PCR amplified donor DNA containing CMV-driven puromycinresistant gene were co-transfected into HEK293T cells. Then cells weretreated with puromycin 7 days after transfection. Single clones wereisolated from puromycin resistant cells followed by verification throughsequencing and Western blot.

RNA Editing of Endogenous or Exogenous-Expressed Transcripts

For assessing RNA editing on the dual fluorescence reporter, HEK293Tcells or HEK293T ADAR1^(−/−) cells were seeded in 6-well plates (6×10⁵cells/well). 24 hours later, cells were co-transfected with 1.5 μgreporter plasmids and 1.5 μg arRNA plasmids. To examine the effect ofADAR1^(p110), ADAR1^(p150) or ADAR2 protein expression, the editingefficiency was assayed by EGFP positive ratio and deep sequencing.

HEK293T ADAR1^(−/−) cells were seeded in 12-well plates (2.5×10⁵cells/well). 24 hours later, cells were co-transfected with 0.5 μg ofreporter plasmids, 0.5 μg arRNA plasmids and 0.5 μg ADAR1/2 plasmids(pLenti backbone as control). The editing efficiency was assayed by EGFPpositive ratio and deep sequencing.

To assess RNA editing on endogenous mRNA transcripts, HEK293T cells wereseeded in 6-well plates (6×10⁵ cells/well). Twenty-four hours later,cells were transfected with 3 μg of arRNA plasmids. The editingefficiency was assayed by deep sequencing.

To assess RNA editing efficiency in multiple cell lines, 8-9×104 (RD,SF268, HeLa) or 1.5×10⁵ (HEK293T) cells were seeded in 12-well plates.For cells difficult to transfect, such as HT29, A549, HepG2, SW13,NIH3T3, MEF and B16, 2-2.5×10⁵ cells were seeded in 6-well plate.Twenty-four hours later, reporters and arRNAs plasmid wereco-transfected into these cells. The editing efficiency was assayed byEGFP positive ratio.

To evaluate EGFP positive ratio, at 48 to 72 hrs post transfection,cells were sorted and collected by Fluorescence-activated cell sorting(FACS) analysis. The mCherry signal was served as a fluorescentselection marker for the reporter/arRNA-expressing cells, and thepercentages of EGFP⁺/mCherry⁺ cells were calculated as the readout forediting efficiency.

For NGS quantification of the A to I editing rate, at 48 to 72 hr posttransfection, cells were sorted and collected by FACS assay and werethen subjected to RNA isolation (TIANGEN, DP420). Then, the total RNAswere reverse-transcribed into cDNA via RT-PCR (TIANGEN, KR103-04), andthe targeted locus was PCR amplified with the corresponding primerslisted in Table 1.

TABLE 1 Name of Primer Sequence (5′--->3′) mCherry-SpeI-Ftataactagtatggtgagcaagggcgaggag (SEQ ID NO: 206) mCherry-BsmBI-R1tatacgtctcatctacagattcttccggcgtgtataccttc (SEQ ID NO: 207) EGFP-BsmBI-F1tatacgtctcatagagatccccggtcgccaccgtgagcaagggcgaggagctg (Reporter-1)(SEQ ID NO: 208) EGFP-AscI-R tataggcgcgccttacttgtacagctcgtccatgcc(SEQ ID NO: 209) mCherry-BsmBI-R2tatacgtctcaaggcgctgcctcctccgccgctgcctcctccgccgctgcctcctccgccctgcagcttgtacagctcgtccatgccgccggtg (SEQ ID NO: 210) EGFP-BsmBI-F2tatacgtctcagcctgctcgcgatgctagagggctctgccagtgagcaagggcgaggagctg(Reporter-2) (SEQ ID NO: 211) LbuCas13-SpeI-Ftataactagtatggtggattacaaggatgacgacgataagatgaaagtgacgaaggtaggaggcatttcg(SEQ ID NO: 212) LbuCas13-AscI-Ratatggcgcgccgttttcagactttttctcttccattttgtattcaaacataatcttcac(SEQ ID NO: 213) hADAR1_(DD)-AscI-FTATAGGCGCGCCAGGCGGAGGAGGCAGCGGCGGAGGAGGCAGCCTCCTCCTCTCAAGGTCCCCAGAAGC(SEQ ID NO: 214) hADAR1_(DD)-SbfI-Rtatacctgcaggctacaccttgcgttttttcttgggtactgggcagagataaaagttcttttcc(SEQ ID NO: 215) Deep-seq-F (Reporter-1) cactccaccggcggcatggacgag(SEQ ID NO: 216) Deep-seq-R (Reporter-1) cacgctgaacttgtggccgtttacgtcg(SEQ ID NO: 217) ADAR1-p150-SpeI-Ftataactagtatgaatccgcggcaggggtattccctcagc (SEQ ID NO: 218)ADAR1-p150-AscI-Rtataggcgcgccctacttatcgtcgtcatccttgtaatctactgggcagagataaaagttcttttcctcctgg(SEQ ID NO: 219) ADAR2-SpeI-F tataactagtatggatatagaagatgaagaaaacatgagttc(SEQ ID NO: 220) ADAR2-AscI-Rtataggcgcgccctacttatcgtcgtcatccttgtaatcgggcgtgagtgagaactggtcctgctcg(SEQ ID NO: 221) ADAR1-p110-SpeI-F tataactagtatggccgagatcaaggagaaaatctgc(SEQ ID NO: 222) ADAR1-p110-AscI-Rtataggcgcgccctacttatcgtcgtcatccttgtaatctactgggcagagataaaagttcttttcctcctgg(SEQ ID NO: 223) KRAS-deep-seq-F cgccatttcggactgggag (SEQ ID NO: 224)KRAS-deep-seq-R agagacaggtttctccatcaattac (SEQ ID NO: 225)PPIB-deep-seq-F gagcccgcgagcaacc (SEQ ID NO: 226) PPIB-deep-seq-Rgcagcaggaagaagacggac (SEQ ID NO: 227) FANCC-deep-seq-F1agaagcagttgaagaccagactc (TAC site) (SEQ ID NO: 228) FANCC-deep-seq-Rggccttcacctggaccatag (TAC site) (SEQ ID NO: 229) FANCC-deep-seq-F2agagaagcagttgaagaccaga (TAC site) (SEQ ID NO: 230) FANCC-deep-seq-R2cggccttcacctggaccata (TAC site) (SEQ ID NO: 231) FANCC-deep-seq-F3cagagaagcagttgaagaccaga (TAC site) (SEQ ID NO: 232) FANCC-deep-seq-R3cggccttcacctggaccata (TAC site) (SEQ ID NO: 233) SMAD4-deep-seq-F1tttgtgaaaggctggggacc (SEQ ID NO: 234) SMAD4-deep-seq-R1acaggattgtattttgtagtccacc (SEQ ID NO: 235) SMAD4-deep-seq-F2aggatgagttttgtgaaaggctg (SEQ ID NO: 236) SMAD4-deep-seq-R2attttgtagtccaccatcctgata (SEQ ID NO: 237) SMAD4-deep-seq-F3gatgagttttgtgaaaggctgg (SEQ ID NO: 238) SMAD4-deep-seq-R3attttgtagtccaccatcctgataa (SEQ ID NO: 239) TRAPPC12-deep-seq-Fcgaagagaacgagaccgcat (SEQ ID NO: 240) TRAPPC12-deep-seq-Rgaagatggtgcacaccggg (SEQ ID NO: 241) TARDBP-deep-seq-Fgacagatgcttcatcagcagtg (SEQ ID NO: 242) TARDBP-deep-seq-Rcgaacaaagccaaaccccttt (SEQ ID NO: 243) COL3A1-deep-seq-Ftctgttaatggacaaatagaaagcc (SEQ ID NO: 244) COL3A1-deep-seq-Rggaacattcaaaggattggcact (SEQ ID NO: 245) BMPR2-deep-seq-Fagtcactgcagatggacgca (SEQ ID NO: 246) BMPR2-deep-seq-Ratctcgatgggaaattgcaggt (SEQ ID NO: 247) AHI1-deep-seq-Ftcagagttttacctcatccttcttt (SEQ ID NO: 248) AHI1-deep-seq-Rcctgaatacatatgatgaccttcag (SEQ ID NO: 249) FANCC-deep-seq-Fagggcacagacacagacctc (Site2) (SEQ ID NO: 250) FANCC-deep-seq-Ragggctttcaatgccaagacg (Site2) (SEQ ID NO: 251) MYBPC3-deep-seq-Ftgacaagccaagtcctccc (SEQ ID NO: 252) MYBPC3-deep-seq-Rattgccaatgatgagctctgg (SEQ ID NO: 253) IL2RG-deep-seq-Fttatagacataagttctccttgcct (SEQ ID NO: 254) IL2RG-deep-seq-Rtcaatcccatggagccaaca (SEQ ID NO: 255) 1-deep-seq-Ftacacgacgctcttccgatcttaagtagaggccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 256) 2-deep-seq-Ftacacgacgctcttccgatctatcatgcttagccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 257) 3-deep-seq-Ftacacgacgctcttccgatctgatgcacatctgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 258) 4-deep-seq-Ftacacgacgctcttccgatctcgattgctcgacgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 259) 5-deep-seq-Ftacacgacgctcttccgatcttcgatagcaattcgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 260) 6-deep-seq-Ftacacgacgctcttccgatctatcgatagttgcttgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 261) 7-deep-seq-Ftacacgacgctcttccgatctgatcgatccagttaggccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 262) 8-deep-seq-Ftacacgacgctcttccgatctcgatcgatttgagcctgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 263) 9-deep-seq-Ftacacgacgctcttccgatctacgatcgatacacgatcgccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 264) 10-deep-seq-Ftacacgacgctcttccgatcttacgatcgatggtccagagccgccactccaccggcggc (Reporter-3)(SEQ ID NO: 265) 1-deep-seq-Ragacgtgtgctcttccgatcttaagtagagtcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 266) 2-deep-seq-Ragacgtgtgctcttccgatctatcatgcttatcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 267) 3-deep-seq-Ragacgtgtgctcttccgatctgatgcacatcttcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 268) 4-deep-seq-Ragacgtgtgctcttccgatctcgattgctcgactcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 269) 5-deep-seq-Ragacgtgtgctcttccgatcttcgatagcaattctcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 270) 6-deep-seq-Ragacgtgtgctcttccgatctatcgatagttgctttcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 271) 7-deep-seq-Ragacgtgtgctcttccgatctgatcgatccagttagtcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 272) 8-deep-seq-Ragacgtgtgctcttccgatctcgatcgatttgagccttcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 273) 9-deep-seq-Ragacgtgtgctcttccgatctacgatcgatacacgatctcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 274) 10-deep-seq-Ragacgtgtgctcttccgatcttacgatcgatggtccagatcgccgtccagctcgaccag (Reporter-3)(SEQ ID NO: 275) ST3GAL1-deep-seq-F ggggaactcgggcaacct (SEQ ID NO: 276)ST3GAL1-deep-seq-R gaatcggatctgccccgtg (SEQ ID NO: 277) EHD2-deep-seq-Fcatcgaggccaagctggaa (SEQ ID NO: 278) EHD2-deep-seq-Rgtagtgaggagggagacccc (SEQ ID NO: 279) OSTM1-AS1-deep-seq-Faagcctccttccttccccaa (SEQ ID NO: 280) OSTM1-AS1-deep-seq-Ratcgatacactccctagccca (SEQ ID NO: 281) IL6-qPCR-F1acaaattcggtacatcctcgac (SEQ ID NO: 282) IL6-qPCR-R1ttcagccatctttggaaggtt (SEQ ID NO: 283) INF-β-qPCR-F1acgccgcattgaccatctat (SEQ ID NO: 284) INF-β-qPCR-R1 tagccaggaggttctcaaca(SEQ ID NO: 285) GAPDH-F1 ggcatggactgtggtcatgag (SEQ ID NO: 286)GAPDH-R1 tgcaccaccaactgcttagc (SEQ ID NO: 287) Reporter-1-qPCR-Fccccgtaatgcagaagaagacc (SEQ ID NO: 288) Reporter-1-qPCR-Rgtccttcagcttcagcctctg (SEQ ID NO: 289) PPIB-qPCR-F aacgcaacatgaaggtgctc(SEQ ID NO: 290) PPIB-qPCR-R accttgacggtgactttggg (SEQ ID NO: 291)KRAS-qPCR-F cagtgcaatgagggaccagt (SEQ ID NO: 292) KRAS-qPCR-Raggaccataggtacatcttcagag (SEQ ID NO: 293) SMAD4-qPCR-Fcgaacgagttgtatcacctgga (SEQ ID NO: 294) SMAD4-qPCR-Rcgatggctgtccctcaaagt (SEQ ID NO: 295) FANCC-qPCR-Fagttgctcttttcactcaaggtc (SEQ ID NO: 296) FANCC-qPCR-Rttctctctgagttcagacgct (SEQ ID NO: 297) PPIB-deep-seq-F (AAGtacacgacgctcttccgatcttaagtagagtggcacaggaggaaagagcatc site)(SEQ ID NO: 298) PPIB-deep-seq-R (AAGagacgtgtgctcttccgatcttaagtagaggcaccacctccatgccctc site) (SEQ ID NO: 299)PPIB-deep-seq-F (CAG tacacgacgctcttccgatcttaagtagagcatcgcagactgcggcaagsite) (SEQ ID NO: 300) PPIB-deep-seq-R (CAGagacgtgtgctcttccgatcttaagtagagagtccatgggcctgtggaatgt site)(SEQ ID NO: 301) FANCC-deep-seq-F2 gaaaaactggcccgagagc (AAG/CAG site)(SEQ ID NO: 302) FANCC-deep-seq-R2 ctgagtctgggctgagggac (AAG/CAG site)(SEQ ID NO: 303) IDUA-deep-seq-F cgcttccaggtcaacaacac (SEQ ID NO: 304)IDUA-deep-seq-R ctcgcgtagatcagcaccg (SEQ ID NO: 305) p53-deep-seq-Fcccctctgagtcaggaaacat (SEQ ID NO: 306) p53-deep-seq-Rgaagatgacaggggccagg (SEQ ID NO: 307) IFN-β-qPCR-F tagcactggctggaatgag(SEQ ID NO: 308) IFN-β-qPCR-R gtttcggaggtaacctgtaag (SEQ ID NO: 309)ISG56-qPCR-F tacagcaaccatgagtacaa (SEQ ID NO: 310) ISG56-qPCR-Rtcaggtgtttcacataggc (SEQ ID NO: 311) ISG54-qPCR-F ctgcaaccatgagtgagaa(SEQ ID NO: 312) ISG54-qPCR-R cctttgaggtgctttagatag (SEQ ID NO: 313)IL-6-qPCR-F gccctgagaaaggagacat (SEQ ID NO: 314) IL-6-qPCR-Rctgttctggaggtactctaggtat (SEQ ID NO: 315) IL-8-qPCR-F tttgaagagggctgagaa(SEQ ID NO: 316) IL-8-qPCR-R tgttctggatatttcatgg (SEQ ID NO: 317)RANTES-qPCR-F catctgcctccccatattcc (SEQ ID NO: 318) RANTES-qPCR-Rtccatcctagctcatctccaaa (SEQ ID NO: 319) IL-12-qPCR-F tgctccagaaggccagac(SEQ ID NO: 320) IL-12-qPCR-R ttcataaatactactaaggcacagg (SEQ ID NO: 321)IL-1β-qPCR-F acagatgaagtgctccttcca (SEQ ID NO: 322) IL-1β-qPCR-Rgtcggagattcgtagctggat (SEQ ID NO: 323) MCP1-qPCR-F cattgtggccaaggagatctg(SEQ ID NO: 324) MCP1-qPCR-R cttcggagtttgggtttgctt (SEQ ID NO: 325)MIP1A-qPCR-F catcacttgctgctgacacg (SEQ ID NO: 326) MIP1A-qPCR-Rtgtggaatctgccgggag (SEQ ID NO: 327) IP10-qPCR-F ctgactctaagtggcatt(SEQ ID NO: 328) IP10-qPCR-R tgatggccttcgattctg (SEQ ID NO: 329)GAPDH-qPCR-F2 cggagtcaacggatttggtcgta (SEQ ID NO: 330) GAPDH-qPCR-R2agccttctccatggtggtgaagac (SEQ ID NO: 331) The PCR products were purifiedfor Sanger sequencing or NGS (Illumina HiSeq X Ten).

Deep Sequencing

For endogenous mRNA editing experiments, 293T-WT cells were seeded on 6wells plates (6×10⁵ cells/well), When approximately 70% confluent,HEK293 cells were transfected with 3 μg dRNA using the X-tremeGENE HPDNA transfection reagent (Roche). 72 hours later, sorted GFP-positive orBFP-positive cells (according to the corresponding fluorescence marker)via FACS, followed by RNA isolation. Then the isolated RNA wasreverse-transcribed into cDNA via RT-PCR, and specific targeted genelocus were amplified with the corresponding primer pairs (23 PCR cycles)and sequenced on an Illumina NextSeq.

Testing in Multiple Cell Lines

Besides HEK293T (positive control) and HEK293T ADAR1^(−/−) (negativecontrol) cells, one mouse cell line (NIH3T3) as well as seven human celllines (RD, HeLa, SF268, A549, HepG2, HT-29, SW13) originating fromdifferent tissues and organs were selected to perform the experiment.For the cell lines with higher transfection efficiency, about 8-9×10⁴cells (RD, HeLa, SF268) or 1.5×10⁵ (HEK293T) were plated onto each wellof 12-well plate, as for the ones (A549, HepG2, HT-29, SW13, NIH3T3)which are difficult to transfect, 2-2.5×10⁵ cells were plated in 6-wellplate. And all these cells were maintained in Dulbecco's modifiedEagle's medium (DMEM, Corning) supplemented with 10% fetal bovine serum(FBS, CellMax) with 5% CO₂ in 37° C. 24 hrs later, CG2 reporter and 71nt dRNA (35-C-35) plasmid were co-transfected into different type ofcells with X-tremeGENE HP DNA transfection reagent (Roche). 48 hrs aftertransfection, cells were trypsinized and analyzed through FACS (BD).Because the cells with low transfection efficiency had quite fewermCherry and BFP positive cells, we increased the total cell number forFACS analysis to 1×10⁵ for those cells plated onto 6-well plate.

RNA Editing Analysis for Targeted Sites

For deep sequencing analysis, an index was generated using the targetedsite sequence (upstream and downstream 20-nt) of arRNA coveringsequences. Reads were aligned and quantified using BWA version0.7.10-r789. Alignment BAMs were then sorted by Samtools, and RNAediting sites were analyzed using REDitools version 1.0.4. Theparameters are as follows: -U [AG or TC]-t 8 -n 0.0 -T 6-6 -e -d -u. Allthe significant A>G conversion within arRNA targeting region calculatedby Fisher's exact test (p-value<0.05) were considered as edits by arRNA.The conversions except for targeted adenosine were off-target edits. Themutations that appeared in control and experimental groupssimultaneously were considered as SNP.

Transcriptome-Wide RNA-Sequencing Analysis

The Ctrl RNA₁₅₁ or arRNA₁₅₁-PPIB-expressing plasmids with BFP expressioncassette were transfected into HEK293T cells. The BFP⁺ cells wereenriched by FACS 48 hours after transfection, and RNAs were purifiedwith RNAprep Pure Micro kit (TIANGEN, DP420). The mRNA was then purifiedusing NEBNext Poly(A) mRNA Magnetic Isolation Module (New EnglandBiolabs, E7490), processed with the NEBNext Ultra II RNA Library PrepKit for Illumina (New England Biolabs, E7770), followed by deepsequencing analysis using Illumina HiSeq X Ten platform (2×150-bp pairedend; 30G for each sample). To exclude nonspecific effect caused bytransfection, we included the mock group in which we only treated cellswith transfection reagent. Each group contained four replications.

The bioinformatics analysis pipeline was referred to the work by Vogelet al²². The quality control of analysis was conducted by using FastQC,and quality trim was based on Cutadapt (the first 6-bp for each readswere trimmed and up to 20-bp were quality trimmed). AWK scripts wereused to filtered out the introduced arRNAs. After trimming, reads withlengths less than 90-nt were filtered out. Subsequently, the filteredreads were mapped to the reference genome (GRCh38-hg38) by STARsoftware⁶¹. We used the GATK Haplotypcaller⁶² to call the variants. Theraw VCF files generated by GATK were filtered and annotated by GATKVariantFiltration, bcftools and ANNOVAR⁶³. The variants in dbSNP, 1000Genome⁶⁴, EVS were filtered out. The shared variants in four replicatesof each group were then selected as the RNA editing sites. The RNAediting level of Mock group was viewed as the background, and the globaltargets of Ctrl RNA₁₅₁ and arRNA₁₅₁-PPIB were obtained by subtractingthe variants in the Mock group.

To assess if LEAPER perturbs natural editing homeostasis, we analyzedthe global editing sites shared by Mock group and arRNA₁₅₁-PPIB group(or Ctrl RNA₁₅₁ group). The differential RNA editing rates at nativeA-to-I editing sites were assessed with Pearson's correlationcoefficient analysis. Pearson correlations of editing rate between Mockgroup and arRNA₁₅₁-PPIB group (or Ctrl RNA₁₅₁ group) were calculated andannotated in FIG. 6.

${p\left( {X,Y} \right)} - \frac{\left. {\left. {E\left\lbrack {X - \mu_{X}} \right.} \right)\left( {Y - \mu_{Y}} \right)} \right\rbrack}{\sigma_{X}\sigma_{Y}}$

X means the editing rate of each site in the Mock group; Y means theediting rate of each site in the Ctrl RNA₁₅₁ group (FIG. 6a ) orarRNA₁₅₁-PPIB group (FIG. 6b ); σ_(x) is the standard deviation of X;σ_(Y) is the standard deviation of Y; μ_(X) is the mean of X; μ_(Y) isthe mean of Y; E is the expectation.

The RNA-Seq data were analysed for the interrogation of possibletranscriptional changes induced by RNA editing events. The analysis oftranscriptome-wide gene expression was performed using HISAT2 andSTRINGTIE software⁶⁵. We used Cutadapt and FastQC for the qualitycontrol of the sequencing data. The sequencing reads were then mapped toreference genome (GRCh38-hg38) using HISAT2, followed by Pearson'scorrelation coefficient analysis as mentioned above.

Western Blot

We used the mouse monoclonal primary antibodies respectively againstADAR1 (Santa Cruz, sc-271854), ADAR2 (Santa Cruz, sc-390995), ADAR3(Santa Cruz, sc-73410), p53 (Santa Cruz, sc-99), KRAS (Sigma,SAB1404011); GAPDH (Santa Cruz, sc-47724) and β-tubulin (CWBiotech,CW0098). The HRP-conjugated goat anti-mouse IgG (H+L, 115-035-003)secondary antibody was purchased from Jackson ImmunoResearch. 2×10⁶cells were sorted to be lysed and an equal amount of each lysate wasloaded for SDS-PAGE. Then, sample proteins were transferred onto PVDFmembrane (Bio-Rad Laboratories) and immunoblotted with primaryantibodies against one of the ADAR enzymes (anti-ADAR1, 1:500;anti-ADAR2, 1:100; anti-ADAR3, 1:800), followed by secondary antibodyincubation (1:10,000) and exposure. The 3-Tubulin was re-probed on thesame PVDF membrane after stripping of the ADAR proteins with thestripping buffer (CWBiotech, CW0056). The experiments were repeatedthree times. The semi-quantitative analysis was done with Image Labsoftware.

Cytokine Expression Assay

HEK293T cells were seeded on 12 wells plates (2×10⁵ cells/well). Whenapproximately 70% confluent, cells were transfected with 1.5 μg ofarRNA. As a positive control, 1 μg of poly(I:C) (Invitrogen, tlrl-picw)was transfected. Forty-eight hours later, cells were collected andsubjected to RNA isolation (TIANGEN, DP430). Then, the total RNAs werereverse-transcribed into cDNA via RT-PCR (TIANGEN, KR103-04), and theexpression of IFN-β and IL-6 were measured by quantitative PCR (TAKARA,RR820A). The sequences of the primers were listed in Table 1.

Transcriptional Regulatory Activity Assay of p53

The TP53^(W53X) cDNA-expressing plasmids and arRNA-expressing plasmidswere co-transfected into HEK293T TP53^(−/−) cells, together withp53-Firefly-luciferase cis-reporting plasmids (YRGene, VXS0446) andRenilla-luciferase plasmids (a gift from Z. Jiang's laboratory, PekingUniversity) for detecting the transcriptional regulatory activity ofp53. 48 hrs later, the cells were harvested and assayed with the PromegaDual-Glo Luciferase Assay System (Promega, E4030) according to themanufacturer protocol. Briefly, 150 μLDual-Glo Luciferase Reagent wasadded to the harvested cell pellet, and 30 minutes later, the Fireflyluminescence was measured by adding 100 μL Dual-Glo Luciferase Reagent(cell lysis) to 96-well white plate by Infinite M200 reader (TECAN). 30min later, 100 μL Dual-Glo stop and Glo Reagent were sequentially addedto each well to measure the Renilla luminescence and calculate the ratioof Firefly luminescence to Renilla luminescence.

Electroporation in Primary Cells

For arRNA-expressing plasmids electroporation in the human primarypulmonary fibroblasts or human primary bronchial epithelial cells, 20 μgplasmids were electroporated with Nucleofector™ 2 b Device (Lonza) andBasic Nucleofector™ Kit (Lonza, VPI-1002), and the electroporationprogram was U-023. For arRNA-expressing plasmids electroporation inhuman primary T cells, 20 μg plasmids were electroporated into humanprimary T with Nucleofector® 2 b Device (Lonza) and Human T cellNucleofector™ Kit (Lonza, VPA-1002), and the electroporation program wasT-024. Forty-eight hours post-electroporation, cells were sorted andcollected by FACS assay and were then subjected to the followingdeep-sequencing for targeted RNA editing assay. The electroporationefficiency was normalized according to the fluorescence marker.

For the chemosynthetic arRNA or control RNA electroporation in humanprimary T cells or primary GM06214 cells, RNA oligo was dissolved in 100μL opti-MEM medium (Gbico, 31985070) with the final concentration 2 μM.Then 1×10E6 GM06214 cells or 3×10E6 T cells were resuspended with theabove electroporation mixture and electroporated with Agile Pulse InVivo device (BTX) at 450 V for 1 ms. Then the cells were transferred towarm culture medium for the following assays.

α-L-Iduronidase (IDUA) Catalytic Activity Assay

The harvested cell pellet was resuspendedand lysed with 28 μL 0.5%Triton X-100 in 1×PBS buffer on ice for 30 minutes. And then 25 μL ofthe cell lysis was added to 25 μL, 190 μM4-methylumbelliferyl-α-L-iduronidase substrate (Cayman, 2A-19543-500),which was dissolved in 0.4 M sodium formate buffer containing 0.2%Triton X-100, pH 3.5, and incubated for 90 minutes at 37° C. in thedark. The catalytic reaction was quenched by adding 200 μL 0.5MNaOH/Glycine buffer, pH 10.3, and then centrifuged for 2 minutes at 4°C. The supernatant was transferred to a 96-well plate, and fluorescencewas measured at 365 nm excitation wavelength and 450 nm emissionwavelength with Infinite M200 reader (TECAN).

Example 1. Testing the RNA Editing Method of the Invention on a Reporter

It has been reported that Cas13 family proteins (C2c2) can edit RNA inmammalian cells. We further tested this system under various conditions.First, we constructed a dual fluorescence reporter system based onmCherry and EGFP fluorescence by introducing 3×GS linker targetingsequence containing stop codon between mCherry and EGFP gene. Inaddition, we deleted the start codon ATG of EGFP in order to reduce theleakage of EGFP translation.

Dual fluorescence reporter-1 comprises sequence of mCherry (SEQ IDNO:1), sequence comprising 3×GS linker and the targeted A (SEQ ID NO:2),and sequence of eGFP (SEQ ID NO:3).

(SEQ ID NO: 1) atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag (sequence of mCherry)(SEQ ID NO: 2) ctgcag

agaaggtatacacgccggaagaatctgtagagatecccggtcgccacc(sequence comprising 3 × GS linker (shown as italicand bold characters) and the targeted A (shown as larger and bold A))(SEQ ID NO: 3) gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatatcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagt aa(sequence of eGFP)

Dual fluorescence reporter-2 comprises sequence of mCherry (SEQ IDNO:1), sequence comprising 3×GS linker (shown as italic and boldcharacters) and the targeted A (shown as larger and bold A) (SEQ IDNO:4), and sequence of eGFP (SEQ ID NO:3).

(SEQ ID NO: 4)) ctgcag

gcctgctcgcgatgctagagggctctgcca(sequence comprising 3 × GS linker (shown as italicand bold characters) and the targeted A (shown aslarger and bold characters))

Dual fluorescence reporter-3 comprises sequence of mCherry (SEQ IDNO:1), sequence comprising 1×GS linker (shown as italic and boldcharacters) and the targeted A (SEQ ID NO:5), and sequence of eGFP (SEQID NO:3).

(SEQ ID NO: 5) ctgcag

gcctgctcgcgatgctagagggctctgcca(sequence comprising 1 × GS linker (shown as italicand bold characters) and the targeted A (shown as larger and bold A))

We cloned mCherry-3×GS linker-TAG-EGFP into pLenti-backbone, and thereporter plasmid was packed into lentivirus, which infected 293T cellsconstructing stable cell line expressing the dual fluorescence reporter.Then, we selected a single clone with low EGFP fluorescence backgroundas the reporter system. We tiled LbucC2c2 crRNA guides with spacers from28 to 78 nucleotides long across the targeting adenosine to test theoptimal crRNA design. We found that longer crRNA guides conferred higherEGFP positive efficiency. Strikingly, when we transfected targetingcrRNA plasmids without co-transfection of any dC2c2-ADARDD-expressingplasmids, the EGFP protein is substantially expressed. For example, thecrRNA guide with the sequence:ggaccaccccaaaaaugaauauaaccaaaacugaacagcuccucgcccuugcucacuggcagagcccuccagcaucgcgagcaggcgcugccuccuccgcc (SEQ ID NO: 6) conferred over 25% EGFP positiveefficiency. This indicates that adenine in the stop codon UAG is largelyedited. In contrast, the random crRNA could not render the EGFP negativecells into positive (FIGS. 6A, 6B and 6C). Based on these results, weinferred that overexpression of a RNA transcript alone could leverageendogenous ADAR enzyme to edit RNA.

Further, we deleted the scaffold RNA sequence on the RNA guides,creating a linear guide RNA. We found 70-nucleotides long RNA(aaaccgagggaucauaggggacugaauccaccauucuucucccaaucccugcaacuccuucuuccccugc(SEQ ID NO: 7)) complementary to the targeting RNA with an A-C mismatchcould efficiently convert the EGFP negative cells into EGFP positivecells, while the 70-nt random RNA(ugaacagcuccucgcccuugcucacuggcagagcccuccagcaucgcgagcaggcgcugccuccuccgcc(SEQ ID NO: 8)) could not (FIGS. 1A, 1B, 1C, and 1D). We thus designatethis RNA as dRNA (Deaminase-recruiting RNA). To verify that the cellularendogenous ADAR could be recruited to conduct adenine deamination bydRNA, we performed experiments in the ADAR1 p110 and ADAR1 p150 doubleknockout 293T cell lines (FIGS. 6E and 6F). Because ADAR1 isubiquitously expressed while ADAR2 is mainly expressed in brain at highlevel. So we proposed the targeting Adenine deamination by dRNA wasmainly mediated by ADAR1 but not ADAR2. As expected, the targeting dRNAcould not trigger EGFP expression in 293T-ADAR1−/− cells, butoverexpressing either exogenous ADAR1 p110, p150 or ADAR2 could rescuethe EGFP expression in 293T-ADAR1−/− cells (FIGS. 1E and 1F), suggestingthat in 293Tcells, the dRNA could recruit ADAR1 or ADAR2 to mediateadenine deamination on a target RNA. Moreover, we found ADAR1-p110 andADAR2 have higher editing activity than ADAR1-p150 (FIG. 1G and FIG.6G), possible due to the different cell localization of ADAR1-p110 andADAR1-p150.

In order to determine the restoration of EGFP fluorescence was due tothe targeting RNA editing events, we directly measured the dRNA-mediatedediting of Reporter-2 transcripts via RT-PCR followed by targeted Sangersequencing and Next-generation sequencing. The sequencing results showedthe A to G base conversion in the targeted Adenine (A-C mismatch site)and the editing rate could reach to 13% (FIG. 6H and FIG. 1H). Besides,we also observed slightly A to G editing during the sequence windowsnear the targeted Adenine, most possibly due to the increased duplex RNAregions, later, we would try to get rid of the unexpected editing withseveral strategies.

Example 2. Optimizing the Factors for Designing dRNAs

Next, we set out to optimize the dRNA to achieve higher editingefficiency. First, we aimed to determine which base in the opposite siteof the targeted adenine favors editing. Previous studies showedtheoppositebaseoftargetedadenosinewouldaffecttheeditingefficiently. Wethus designed 71 nt dRNAs with a mismatch N (A, U, C and G) in themiddle position opposite to targeted A. Based on the FACS results, wefound that the four different dRNAs editing efficiently as follow:C>A>U>G (FIGS. 2A and 2B). Recently, it has been reported that littlebubble in the target UAG site may be of benefit to the editingefficiency. Therefore, we designed dRNAs containing two or threemismatch bases with target UAG site to test our hypothesis. 16 different71 nt dRNAs were designed and constructed on the dRNA vector with BFPmarker using Golden Gate cloning method. We found that the dRNAs withCCA and GCA sequence are of the highest efficiency, which means thelittle bubble contribute little to A-I editing, at least in the case ofUAG target site. Besides, four dRNAs of NCA sequence have higherpercentage of GFP positive cells, leading to the conclusion thatcomplementary U-A base pair may be important for ADAR editing (FIGS. 2Cand 2D). Subsequently, we test the efficiency of different length ofdRNA based on Reporter. dRNAs were designed a mismatch C in the middleposition with different length ranging from 31 nt to 221 nt. We foundthat editing efficiency increases with longer dRNA. The peak of editingof reporter system is located at 171 nt dRNA. 51 ntdRNA could light upreporter system with a good efficiency (18%) (FIGS. 2E and 2F). Finally,we examined whether the position of mismatch C of dRNA affect theediting efficiency. dRNAs were kept the same 71 nt length, a mismatch Cin different position from transcription beginning was designed. Basedon the FACS results, we found that the location of the opposite mismatchC could affect the editing efficiency, and the mismatch C located in the5′ or 3′ of dRNA has a lower efficiency (FIGS. 2G and 2H).

16 different reporter comprising target sequences containing allpossible 3 base motifs were constructed through Gibson cloning, and thencloned into pLenti backbone (pLenti-CMV-MCS-SV-Bsd, Stanley Cohen Lab,Stanford University). The target sequences are shown as follows.

Target sequences containing all possible 3 base motifs:

TAT: (SEQ ID NO: 9) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgctatagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc TAA:(SEQ ID NO: 10) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgctaaagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc TAC:(SEQ ID NO: 11) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgctacagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc TAG:(SEQ ID NO: 12) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgctagagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc AAT:(SEQ ID NO: 13) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcaatagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc AAA:(SEQ ID NO: 14) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcaaaagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc AAC:(SEQ ID NO: 15) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcaacagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc AAG:(SEQ ID NO: 16) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcaagagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc CAT:(SEQ ID NO: 17) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgccatagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc CAA:(SEQ ID NO: 18) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgccaaagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc CAC:(SEQ ID NO: 19) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgccacagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc CAG:(SEQ ID NO: 20) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgccagagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc GAT:(SEQ ID NO: 21) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcgatagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc GAA:(SEQ ID NO: 22) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcgaaagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc GAC:(SEQ ID NO: 23) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcgacagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc GAG:(SEQ ID NO: 24) atggacgagctgtacaagctgcagggcggaggaggcagcgcctgctcgcgatgcgagagggctctgccagtgagcaagggcgaggagctgttcaccggggtg gtgcccatc

dRNAs were kept same 111 bp length and designed a mismatch C at thecenter towards the target A.

In 12-well cell culture cluster, 2×10⁵ cells HEK293T were plated to theeach well and each experiment was performed for three replicates. 24 hrslater, 0.5 μg dRNA plasmid and 0.5 μg reporter target plasmid wereco-transfected to the cells using the X-tremeGENE HP DNA transfectionreagent (Roche). 48 hrs later, cells were trypsinized and selected formCherry positive cells through FACS (BD). A total of 4×10⁵ cells wereharvested and total RNA was extracted using RNAprep pure Cell/BacteriaKit (TIANGEN DP430). The cDNAs were synthesized from 2 μg of total RNAusing Quantscript RT Kit (TIANGEN KR103-04). And the 111 target regionswere amplified through PCR and sent for deep sequencing.

We found that all 16 different 3 base motifs can be edited through anexemplary RNA editing method of the present application, albeit with avariable efficiency. In sum, the results indicate the 5′ nearestneighbor of A to be edited has the preference U>C≈A>G and 3′ nearestneighbor of A to be edited has the preference G>C>A≈U. Data werepresented as bar chart in FIG. 3A or heatmap of FIG. 3B.

Example 3. Editing RNA Transcribed from Endogenous Genes

Next, we tested whether dRNA could mediate mRNA transcribed fromendogenous genes. We designed dRNA targeting four genes KRAS, PPIB,β-Actin and GAPDH. For KRAS mRNA, we designed 91, 111, 131, 151, 171 and191 nucleotides long dRNAs (FIG. 4A) with sequences as shown below.

91-nt KRAS-dRNA (SEQ ID NO: 25)uagcuguaucgucaaggcacucuugccuacgccaccagcuccaaccaccacaaguuuauauucagucauuuucagcaggccucucucccgc 111-nt KRAS-dRNA(SEQ ID NO: 26) gauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaacuaccacaaguuuauauucagucauuuucagcaggccucucucccgca ccugggagc 131-nt KRAS-dRNA (SEQ ID NO: 27)uccacaaaaugauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaacuaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagccgcugagccu 151-nt KRAS-dRNA (SEQ ID NO: 28)aucauauucguccacaaaaugauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaaccaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagccgcugagccucuggccccgc 171-nt KRAS-dRNA(SEQ ID NO: 29) cuauuguuggaucauauucguccacaaaaugauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaaccaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagccgcugagccucu ggccccgccgccgccuuc191-nt KRAS-dRNA (SEQ ID NO: 30)uaggaauccucuauuguuggaucauauucguccacaaaaugauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaaccaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagccgcugagccucuggccccgccgccgccuucagugccugcgThe Next-generation sequencing results showed that the dRNAs could editthe targeted KRAS mRNA with up to 11.7% editing efficiency (FIG. 4B).For endogenous PPIB mRNA, the targeted three sites: site1, site2 andsite3. We designed 151 nucleotides long dRNA for each site (FIG. 4C)with sequences as shown below.

151-nt PPIB-dRNA (site 1) (SEQ ID NO: 31)gaggcgcagcauccacaggcggaggcgaaagcagcccggacagcugaggccggaagaggguggggccgcgguggccagggagccggcgccgccacgcgcgggugggggggacugggguugcucgcgggcuccgggcgggcggcgggcgccg151-nt PPIB-dRNA (site 2) (SEQ ID NO: 32)uccuguagcuaaggccacaaaauuauccacuguuuuuggaacagucuuuccgaagagaccaaagaucacccggcccacaucuucaucuccaauucguaggucaaaauacaccuugacggugacuuugggccccuucuucuucucaucggcc151-nt PPIB-dRNA (site 3) (SEQ ID NO: 33)gcccuggaucaugaaguccuugauuacacgauggaauuugcuguuuuuguagccaaauccuuucucuccuguagccaaggccacaaaauuauccacuguuuuuggaacagucuuuccgaagagaccaaagaucacccggccuacaucuuca

The Next-generation sequencing results showed that the dRNA could editPPIB mRNA site1 efficiently with up to 14% editing rate (FIG. 4D). ForPPIB mRNA site2 and site3, the editing efficiency was 1.5% and 0.6%(FIGS. 4E and 4F). For endogenous β-Actin mRNA, we selected two targetedsite and designed dRNA for each site (FIG. 4G) with sequences as shownbelow.

72-nt β-Actin-dRNA (site 1) (SEQ ID NO: 34)gcgcaaguuagguuuugucaagaaaggguguaacgcaaccaagucauaguccgccuagaagcauuugcggug 131-nt β-Actin-dRNA (site 1) (SEQ ID NO: 35)gccaugccaaucucaucuuguuuucugcgcaaguuagguuuugucaagaaaggguguaacgcaaccaagucauaguccgccuagaagcauuugcgguggacgauggaggggccggacucgucauacuccug 70-nt β-Actin-dRNA (site 2)(SEQ ID NO: 36) ggacuuccuguaacaacgcaucucauauuuggaaugaccauuaaaaaaacaacaaugugcaaucaaagucWe found that dRNA could edit β-Actin mRNA both site1 and site2, with upto 1.4% editing efficiency for each site (FIG. 4H and FIG. 8A). We alsoobserved longer dRNA conferred higher editing efficiency, with 0.6% fordRNA-71 nt and 1.4% for dRNA-131 nt (FIG. 3H). For another housekeepinggene GAPDH, we used 71 nt dRNA(caaggugcggcuccggccccuccccucuucaagggguccacauggcaacugugaggaggggagauucagug(SEQ ID NO: 37)), and the editing efficiency is 0.3%, may be due to theshort dRNA length (FIG. 8B).

Example 4. Off-Targeting Analysis on an Exemplary LEAPER Method

For therapeutic application, the precision of editing is pivotal. Next,we tried to characterize the specificity of an exemplary RNA editingsystem of the present application. We selected endogenous PPIB site1 andKRAS site for analysis. For PPIB site1, we could see during the dRNAcovered regions, there were several A bases flanking the targeted A76,such as A22, A30, A33, A34, A39, A49, A80, A91, A107 and A140. Itrevealed that those flanking A bases were barely edited, while thetargeted A76 base (A-C mismatch) showed up to 14% editing efficiency(FIGS. 5A and 5B).

As for KRAS site, we could see in the dRNA covered region, there aremany adenines flanking the targeted A56 base, up to 29 flanking A bases.From the KRAS mRNA editing results, we found that while the targeted A56base (A-C mismatch) showed up to 11.7% editing efficiency, the flankingadenine could be edited (FIGS. 5C and 5D). A variety of the off-targetedadenines were edited, while adenines such as A41, A43, A45, A46, A74,A79 showed more editing. We found the 5′ nearest neighbor of thoseunedited A bases were G or C, whereas the 5′ nearest neighbor of thoseefficiently edited adenines was T or A. Based on this observation, weset out to design dRNA to minimize the off-target editing of thoseadenines that are prone to be edited. In our study, we have found ADARpreferred A-C mismatch to A-A, A-U, and, the A-G mismatch was the leastpreferred. So, we proposed that for the off-targeting A bases to whichthe 5′ nearest neighbor was U or A, A-G mismatch might reduce ordiminish the off-targeting effects. Previous study has reported A-Gmismatch could block the deamination editing by ADAR.

So next we designed three kinds of 91-nt dRNA variants and four kinds of111-nt dRNA variants (with sequences as shown below) containingdifferent A-G mismatch combinations based on the statistical results inFIG. 5D and existing knowledge: dRNA-AG1 (A41, A46, A74); dRNA-AG2 (A41,A43, A45, A46, A74, A79); dRNA-AG3 (A31, A32, A33, A41, A43, A45, A46,A47, A74, A79); dRNA-AG4 (A7, A31, A32, A33, A40, A41, A43, A45, A46,A47, A74, A79, A95) (FIG. 4E).

KRAS-dRNA-91-AG2 (SEQ ID NO: 38)UAGCUGUAUCGUCAAGGCACUCgUGCCgACGCCACCAGCUCCAACcACCACAAGgggAgAgUCAGUCAgggUCAGCAGGCCUCUCUCCCGC KRAS-dRNA-91-AG3(SEQ ID NO: 39) UAGCUGUAUCGUCAAGGCACUCUUGCCgACGCCACCAGCUCCAACcACCACAAGUgUAUAgUCAGUCAUUUUCAGCAGGCCUCUCUCCCGC KRAS-dRNA-91-AG4(SEQ ID NO: 40) UAGCUGGAUCGUCAAGGCACUCGUGCCGACGCCACCAGCUCCAACCACCACAAGGGGAGAGGCAGUCAGGGUCAGCAGGCCUCUCUCCCGC KRAS-dRNA-111-AG1(SEQ ID NO: 41) GAUUCUGAAUUAGCUGUAUCGUCAAGGCACUCUUGCCgACGCCACCAGCUCCAACcACCACAAGUgUAUAgUCAGUCAUUUUCAGCAGGCCUCUCUCCCGCA CCUGGGAGCKRAS-dRNA-111-AG2 (SEQ ID NO: 42)GAUUCUGAAUUAGCUGUAUCGUCAAGGCACUCgUGCCgACGCCACCAGCUCCAACcACCACAAGUggAgAgUCAGUCAUUUUCAGCAGGCCUCUCUCCCGCA CCUGGGAGCKRAS-dRNA-111-AG3 (SEQ ID NO: 43)GAUUCUGAAUUAGCUGUAUCGUCAAGGCACUCgUGCCgACGCCACCAGCUCCAACcACCACAAGgggAgAgUCAGUCAgggUCAGCAGGCCUCUCUCCCGCA CCUGGGAGCKRAS-dRNA-111-AG4 (SEQ ID NO: 44)GCUCCCCGGUGCGGGAGAGAGGCCUGCUGACCCUGACUGCCUCUCCCCUUGUGGUGGUUGGAGCUGGUGGCGUCGGCACGAGUGCCUUGACGAUCCAGCUAA UUCAGAAUC

Then these dRNAs were transfected into HEK293T cells, and empty vectorand 71-nt non-targeting dRNA control:(tctcagtccaatgtatggtccgagcacaagctctaatcaaagtccgcgggtgtagaccggttgccatagga(SEQ ID NO: 45)) were used as negative controls. For 91-nt dRNAs, thedeep sequencing results showed that the on-target editing (A56) wasreduced to 2.8% for dRNA-91-AG2, 2.3% for dRNA-91-AG3 and 0.7% fordRNA-91-AG4, compared to the on-target editing (A56) efficiency 7.9% fordRNA-91 without A-G mismatch (FIG. 4F). For 91-nt dRNAs, the on-targetediting (A56) was reduced to 5.1% for dRNA-111-AG2 and 4.9% fordRNA-111-AG3 compared to the on-target editing (A56) efficiency 15.1%for dRNA-111 without A-G mismatch (FIG. 4F), which indicating longerdRNA could bear more A-G mismatch. So next we selected 111-nt dRNA fordetailed off-target analysis. The flanking A bases editing were wipedout dramatically except for A7 and A79 (FIG. 4G). For A7 base, theoff-target effect could be prevented by a further A-G mismatch design atthis site, which is absent in the current dRNA design. For A79 base,introducing adjacent two A-G mismatch A78/A79 might help to wipe out theoff-target effects. Based on such results, applying the RNA editingsystems of the present application to cure genetic diseases is verypromising and encouraging.

Example 5. Testing an Exemplary LEAPER Method in Multiple Cell Lines

Through the results in HEK293T cells, we supposed that the double strandRNA formed by linear dRNA and its target RNA could recruit endogenousADAR protein for A-I editing. To confirm the hypothesis, we chose morecell lines to test our RNA editing method. The results are shown in FIG.9. Those results in multiple cell lines proved the universality of ourRNA editing method. Firstly, despite of the various editing efficiency,using dRNA to recruit endogenous ADAR was suitable for multiple humancell lines, which was originated from 7 different tissues and organs.Furthermore, this method could not only work in human cells, but also inmouse cells, providing the possibility to conduct experiments on amouse.

Example 6. Leveraging Endogenous ADAR for RNA Editing

In an attempt to explore an efficient RNA editing platform, we fused thedeaminase domain of the hyperactive E1008Q mutant ADAR1 (ADAR1_(DD))⁴⁰to the catalytic inactive LbuCas13 (dCas13a), an RNA-guidedRNA-targeting CRISPR effector⁴¹ (FIG. 10A). To assess RNA editingefficiency, we constructed a surrogate reporter harbouring mCherry andEGFP genes linked by a sequence comprising a 3×GGGGS-coding region andan in-frame UAG stop codon (Reporter-1, FIG. 10B). Thereporter-transfected cells only expressed mCherry protein, whiletargeted editing on the UAG of the reporter transcript could convert thestop codon to UIG and consequently permit the downstream EGFPexpression. Such a reporter allows us to measure the A-to-I editingefficiency through monitoring EGFP level. We then designed hU6promoter-driven crRNAs (CRISPR RNAs) containing 5′ scaffolds subjectedfor Cas13a recognition and variable lengths of spacer sequences fortargeting (crRNA^(Cas13a), following LbuCas13 crRNA sequences).

TABLE 2 LbuCas13 crRNA sequences Name Sequence Source LbuCas13/Cas13aggaccaccccaaaaaugaaggggacuaaaac FIG. 10 crRNA scaffold (SEQ ID NO: 46)Ctrl crRNA₇₀aaaccgagggaucauaggggacugaauccaccauucuucucccaaucccugcaacuccuucuuccccugcFIG. 10 (SEQ ID NO: 47) Spacer of crRNA₁₅ gcagagccucCagc FIG. 10(SEQ ID NO: 48) Spacer of crRNA₂₂ cucacuggcagagccucCagc FIG. 10(SEQ ID NO: 49) Spacer of crRNA₂₈ cccuugcucacuggcagagccucCagc FIG. 10(SEQ ID NO: 50) Spacer of crRNA₃₅ cucucgcccuugcucacuggcagagccucCagcFIG. 10 (SEQ ID NO: 51) Spacer of crRNA₄₀cucucgcccuugcucacuggcagagccucCagcaucgc FIG. 10 (SEQ ID NO: 52)Spacer of crRNA₄₇ ugaacagcucucgcccuugcucacuggcagagccucCagcaucgc FIG. 10(SEQ ID NO: 53) Spacer of crRNA₇₀ugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccFIG. 10 (SEQ ID NO: 54)

The sequences complementary to the target transcripts all contain CCAopposite to the UAG codon so as to introduce a cytidine (C) mis-pairingwith the adenosine (A) (FIG. 10B) because adenosine deaminationpreferentially occurs in the A-C mismatch site^(13, 14). To test theoptimal length of the crRNA, non-targeting or targeting crRNAs ofdifferent lengths were co-expressed with dCas13a-ADAR1_(DD) proteins inHEK293T cells stably expressing the Reporter-1. Evident RNA editingeffects indicated by the appearance of EGFP expression were observedwith crRNAs containing matching sequences at least 40-nt long, and thelonger the crRNAs the higher the EGFP positive percentage (FIG. 10C).Surprisingly, expression of long crRNA^(Cas13a) alone appearedsufficient to activate EGFP expression, and the co-expression ofdCas13a-ADAR1_(DD) rather decreased crRNA activity (FIGS. 10C, 10D). TheEGFP expression was clearly sequence-dependent because the 70-nt(exclusive of the 5′ scaffold for the length calculation) control RNAcould not activate EGFP expression (FIGS. 10C, 10D).

With the surprising finding that certain long engineered crRNA^(Cas13a)enabled RNA editing independent of dCas13a-ADAR1_(DD), we decided toremove the Cas13a-recruiting scaffold sequence from the crRNA. Becausethe crRNA₇₀ had the highest activity to trigger EGFP expression (FIG.10C, 10D), we chose the same 70-nt long guide RNA without theCas13a-recruiting scaffold for further test (FIG. 11A and the Sequencesof arRNAs in Table 3 and control RNAs used in the examples).

TABLE 3 Name Sequence (5′ ---> 3′) Source Ctrl RNA₇₀AaaccgagggaucauaggggacugaauccaccauucuucucccaaucccugcaacuccuucuuccccugcFIG. 11 (SEQ ID NO: 55) arRNA₇₀ugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgcc(SEQ ID NO: 56) Ctrl RNA₇₁UcucaguccaauguaugguccgagcacaagcucuaaucaaaguccgcggguguagaccgguugccauaggaFIG. 14 (SEQ ID NO: 57) and arRNA₇₁acagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugFIG. 16 (SEQ ID NO: 58) arRNA₇₁-CAAacagcuccucgcccuugcucacuggcagagcccucAagcaucgcgagcaggcgcugccuccuccgccgcugFIG. 16A (SEQ ID NO: 59) arRNA₇₁-CUAacagcuccucgcccuugcucacuggcagagcccucUagcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 60) arRNA₇₁-CGAacagcuccucgcccuugcucacuggcagagcccucGagcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 61) arRNA₇₁-GCAacagcuccucgcccuugcucacuggcagagcccuGCAgcaucgcgagcaggcgcugccuccuccgccgcugFIG. 16B, (SEQ ID NO: 62) C arRNA₇₁-UCAacagcuccucgcccuugcucacuggcagagcccuUCAgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 63) arRNA₇₁-ACAacagcuccucgcccuugcucacuggcagagcccuACAgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 64) arRNA₇₁-CCUacagcuccucgcccuugcucacuggcagagcccuCCUgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 65) arRNA₇₁-GCUacagcuccucgcccuugcucacuggcagagcccuGCUgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 66) arRNA₇₁-UCUacagcuccucgcccuugcucacuggcagagcccuUCUgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 67) arRNA₇₁-ACUacagcuccucgcccuugcucacuggcagagcccuACUgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 68) arRNA₇₁-CCCacagcuccucgcccuugcucacuggcagagcccuCCCgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 69) arRNA₇₁-GCCacagcuccucgcccuugcucacuggcagagcccuGCCgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 70) arRNA₇₁-UCCacagcuccucgcccuugcucacuggcagagcccuUCCgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 71) arRNA₇₁-ACCacagcuccucgcccuugcucacuggcagagcccuACCgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 72) arRNA₇₁-CCGacagcuccucgcccuugcucacuggcagagcccuCCGgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 73) arRNA₇₁-GCGacagcuccucgcccuugcucacuggcagagcccuGCUgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 74) arRNA₇₁-UCGacagcuccucgcccuugcucacuggcagagcccuUCGgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 75) arRNA₇₁-ACGacagcuccucgcccuugcucacuggcagagcccuACGgcaucgcgagcaggcgcugccuccuccgccgcug(SEQ ID NO: 76) arRNA₃₁- acuggcagagcccucCagcaucgcgagcagg FIG. 16DReporter-1 (SEQ ID NO: 77) and arRNA₅₁-gcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccucc FIG. 27 Reporter-1(SEQ ID NO: 78) arRNA₉₁-acagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugReporter-1 (SEQ ID NO: 79) arRNA₁₁₁-acccCggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccReporter-1 uccgccgcugccuccuccgc (SEQ ID NO: 80) arRNA₁₃₁-gcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcauReporter-1 cgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugc(SEQ ID NO: 81) arRNA₁₅₁-ucgccguccagcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagccReporter-1cucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguaca (SEQ ID NO: 82) arRNA₁₇₁-gccguuuacgucgccguccagcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacReporter-1uggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguacagcucguccau (SEQ ID NO: 83) arRNA₁₉₁-ugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccaccccggugaacagcuccucgcReporter-1ccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguacagcucguccaugccgccggug (SEQ ID NO: 84)arRNA₂₁₁-ccggacacgcugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccaccccggugaacReporter-1agcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguacagcucguccaugccgccgguggaguggcggc(SEQ ID NO: 85) arRNA₃₁- gcgaccggggaucucCacagauucuuccggc Reporter-2(SEQ ID NO: 86) arRNA₅₁-gcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccu Reporter-2(SEQ ID NO: 87) arRNA₇₁-ccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuReporter-2 (SEQ ID NO: 88) arRNA₉₁-gugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuuReporter-2 cugcugccuccuccgccgc (SEQ ID NO: 89) arRNA₁₁₁-caccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcgReporter-2 uguauaccuucugcugccuccuccgccgcugccuccucc (SEQ ID NO: 90)arRNA₁₃₁-ccaggaugggcaccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauReporter-2 ucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccu(SEQ ID NO: 91) arRNA₁₅₁-uccagcucgaccaggaugggcaccaccceggugaacagcuccucgcccuugcucacgguggcgaccggggauReporter-2cucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccu (SEQ ID NO: 92) arRNA₁₇₁-cggcgacguauccagcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacgguggcReporter-2gaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuugua (SEQ ID NO: 93) arRNA₁₉₁-uguggccguuuacgucgccguccagcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcReporter-2ucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguacagcucgucc (SEQ ID NO: 94)arRNA₂₁₁-acgcugaacuuguggccguuuacgucgccguccagcucgaccaggaugggcaccaccccggugaacagcuccReporter-2ucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuguacagcucguccaugccgccgg(SEQ ID NO: 95) arRNA₇₁(C + 70)-CagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugcagcuuFIG. 16E Reporter-1 (SEQ ID NO: 96) arRNA₇₁(5 + C +cccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgcccugc65)-Reporter-1 (SEQ ID NO: 97) arRNA₇₁(10 + C +cagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccuccuccgc60)-Reporter-1 (SEQ ID NO: 98) arRNA₇₁(15 + C +acuggcagagcccuccCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcugccucc55)-Reporter 1 (SEQ ID NO: 99) arRNA₇₁(20 + C +ugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgccgcug50)-Reporter-1 (SEQ ID NO: 100) arRNA₇₁(25 + C +gcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccuccuccgc45)-Reporter-1 (SEQ ID NO: 101) arRNA₇₁(30 + C +uccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgccgcugccucc40)-Reporter-1 (SEQ ID NO: 102) arRNA₇₁(40 + C +ggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccuccuccgc30)-Reporter-1 (SEQ ID NO: 103) arRNA₇₁(45 + C +accccggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcugccucc25)-Reporter-1 (SEQ ID NO: 104) arRNA₇₁(50 + C +gcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcgcug20)-Reporter-1 (SEQ ID NO: 105) arRNA₇₁(55 + C +gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcagg15)-Reporter-1 (SEQ ID NO: 106) arRNA₇₁(60 + C +accaggaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcga10)-Reporter-1 (SEQ ID NO: 107) arRNA₇₁(65 + C +gcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcau5)-Reporter-1 (SEQ ID NO: 108) arRNA₇₁(70 + C)-guccagcucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCReporter-1 (SEQ ID NO: 109) arRNA₇₁(C + 70)-CacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccuccuccReporter-2 (SEQ ID NO: 110) arRNA₇₁(5 + C +aucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgcugccu65)-Reporter-2 (SEQ ID NO: 111) arRNA₇₁(10 + C +cggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccuccgccgc60)-Reporter-2 (SEQ ID NO: 112) arRNA₇₁(15 + C +gcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccuccucc55)-Reporter-2 (SEQ ID NO: 113) arRNA₇₁(20 + C +cgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgcugccu50)-Reporter-2 (SEQ ID NO: 114) arRNA₇₁(25 + C +gcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccuccgccgc45)-Reporter-2 (SEQ ID NO: 115) arRNA₇₁(30 + C +cccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugcugccuccucc40)-Reporter-2 (SEQ ID NO: 116) arRNA₇₁(40 + C +cagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccuucugc30)-Reporter-2 (SEQ ID NO: 117) arRNA₇₁(45 + C +gugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcguguauaccu25)-Reporter-2 (SEQ ID NO: 118) arRNA₇₁(50 + C +ccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggcgugua20)-Reporter-2 (SEQ ID NO: 119) arRNA₇₁(55 + C +caccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuuccggc15)-Reporter-2 (SEQ ID NO: 120) arRNA₇₁(60 + C +augggcaccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacagauucuu10)-Reporter-2 (SEQ ID NO: 121) arRNA₇₁(65 + C +ccaggaugggcaccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCacaga5)-Reporter-2 (SEQ ID NO: 122) arRNA₇₁(70 + C)-cucgaccaggaugggcaccaccccggugaacagcuccucgcccuugcucacgguggcgaccggggaucucCReporter-2 (SEQ ID NO: 123) arRNA₁₁₁-CCA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCagcaucgcgagcaggcFIG. 16F, Reporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau G(SEQ ID NO: 124) arRNA₁₁₁-GCA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugCagcaucgcgagcaggcReporter-3 (UAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 125) arRNA₁₁₁-UCA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuCagcaucgcgagcaggcReporter-3 (UAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 126) arRNA₁₁₁-ACA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaCagcaucgcgagcaggcReporter-3 (UAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 127) arRNA₁₁₁-CCG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCggcaucgcgagcaggcReporter-3 (CAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 128) arRNA₁₁₁-GCG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugCggcaucgcgagcaggcReporter-3 (CAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 129) arRNA₁₁₁-UCG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuCggcaucgcgagcaggcReporter-3 (CAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 130) arRNA₁₁₁-ACG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaCggcaucgcgagcaggcReporter-3 (CAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 131) arRNA₁₁₁-CCU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 132) arRNA₁₁₁-GCU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugCugcaucgcgagcaggcReporter-3 (AAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 133) arRNA₁₁₁-ACU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaCugcaucgcgagcaggcReporter-3 (AAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 134) arRNA₁₁₁-UCU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuCugcaucgcgagcaggcReporter-3 (AAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 135) arRNA₁₁₁-CCC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucCcgcaucgcgagcaggcReporter-3 (GAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 136) arRNA₁₁₁-GCC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugCcgcaucgcgagcaggcReporter-3 (GAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 137) arRNA₁₁₁-UCC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuCcgcaucgcgagcaggcReporter-3 (GAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 138) arRNA₁₁₁-ACC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaCcgcaucgcgagcaggcReporter-3 (GAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 139) Ctrl RNA₁₁₁UaccgcuacagccacgcugauuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucucuFIG. 17 gcuggggaauugcgcgauauucaggauuaaaagaagugc (SEQ ID NO: 140)Ctrl RNA₁₅₁Acuacaguugcuccgauauuuaggcuacgucaauaggcacuaacuuauuggcgcuggugaacggacuuccucucgaguaccagaagaugacuacaaaacuccuuuccauugcgaguaucggagucuggcucaguuuggccagggaggcacu (SEQ ID NO: 141) arRNA₅₁-PPIBcggaagaggguggggccgcgguggcCagggagccggcgccgccacgcgcgg FIG. 17B(SEQ ID NO: 142) arRNA₇₁-PPIBcagcugaggccggaagaggguggggccgcgguggcCagggagccggcgccgccacgcgcggguggggggga(SEQ ID NO: 143) arRNA₁₁₁-PPIBggaggcgaaagcagcccggacagcugaggccggaagagggruggggccgcgguggcCagggagccggcgccgccacgcgcgggugggggggacugggguugcucgcgggcuc (SEQ ID NO: 144) arRNA₁₅₁-PPIBgaggcgcagcauccacaggcggaggcgaaagcagcccggacagcugaggccggaagaggguggggccgcgguggcCagggagccggcgccgccacgcgeggsrugggggggacugggguugcucgegggcuccgggegggcggcgggcgccg (SEQ ID NO: 145) arRNA₅₁-KRASucuugccuacgccaccagcuccaacCaccacaaguuuauauucagucauuu (SEQ ID NO: 146)arRNA₇₁-KRASgucaaggcacucuugccuacgccaccagcuccaacCaccacaaguuuauauucagucauuuucagcaggccarRNA₁₁₁-KRASGauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaacCaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagc (SEQ ID NO: 147) arRNA₁₅₁-KRASaucauauucguccacaaaaugauucugaauuagcuguaucgucaaggcacucuugccuacgccaccagcuccaacCaccacaaguuuauauucagucauuuucagcaggccucucucccgcaccugggagccgcugagccucuggccccgc (SEQ ID NO: 148) arRNA₅₁-SMAD4ucggcaugguaugaaguacuucgucCaggagcuggagggcccgguguaagu (SEQ ID NO: 149)arRNA₇₁-SMAD4gggucugcaaucggcaugguaugaaguacuucgucCaggagcuggagggcccgguguaagugaauuucaau(SEQ ID NO: 150) arRNA₁₁₁-SMAD4gaccucagucuaaagguugugggucugcaaucggcaugguaugaaguacuucgucCaggagcuggagggcccgguguaagugaauuucaauccagcaagguguuucuuuga (SEQ ID NO: 151) arRNA₁₅₁-SMAD4uaagggccccaacgguaaaagaccucagucuaaagguugugggucugcaaucggcaugguaugaaguacuucgucCaggagcuggagggcccgguguaagugaauuucaauccagcaagguguuucuuugaugcucugucuuggguaaucc (SEQ ID NO: 152) arRNA₅₁-FANCCugggggguucggcugccgacaucagCaauugcucugccaccaucucagccc (TAC site)(SEQ ID NO: 153) arRNA₇₁-FANCCagcagggccgugggggguucggcugccgacaucagCaauugcucugccaccaucucagcccauccuccgaa(TAC site) (SEQ ID NO: 154) arRNA₁₁₁-FANCCaguagaaggccaagagccacagcagggccgugggggguucggcugccgacaucagCaauugcucugccacca(TAC site) ucucagcccauccuccgaagugaaugaacaggaaccagc (SEQ ID NO: 155)arRNA₁₅₁-FANCCccucccaucacgggggccguaguagaaggccaagagccacagcagggccgugggggguucggcugccgacau(TAC site)cagCaauugcucugccaccaucucagcccauccuccgaagugaaugaacaggaaccagcucucaaagggaccuccgcag (SEQ ID NO: 156) arRNA₁₅₁-PPIBgccaaacaccacatgcttgccatctagccaggctgtcttgactgtcgtgatgaagaactgggagccgttggtFIG. 17C (AAG site)gtcCttgcctgcgttggccatgctcacccagccaggcccgtagtgcttcagtttgaagttctcatcggggaaFIG. 17D gcgctca (SEQ ID NO: 157) arRNA₁₅₁-PPIBgggagtgggtccgctccaccagatgccagcaccggggccagtgcagctcagagccctgtggcggactacagg(CAG site)gccCgcacagacggtcactcaaagaaagatgtccctgtgccctactccttggcgatggcaaagggcttctccacctcga (SEQ ID NO: 158) arRNA₁₅₁-FANCCtgcattttgtaaaatagatactagcagattgtcccaagatgtgtacagctcattctcacagcccagcgaggg(AAG site)acacCtctccacaaatgcgtggccacaggtcatcacctgtectgtggccctggcgagectgatccctcacgccgggcac (SEQ ID NO: 159) arRNA₁₅₁-FANCCgctcattctcacagcccagcgagggcacttactccacaaatgcgtggccacaggtcatcacctgtcctgtgg(CAG site)cccCggcgagcctgatccctcacgccgggcacccacacggcctgcgtgccttctagacttgagttcgcagctattaag (SEQ ID NO: 160) arRNA₁₅₁-IDUAtcggccgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccg(CAG site)gccCgcgacacttcggcccagagctgctectcatccagcagcgccagcagccccatggccgtgagcaccggcttgcgca (SEQ ID NO: 161) arRNA₁₁₁-TARDBPugaccagucuuaagaucuuucuugaccugcaccauaagaacuucuccaaagguacCaaaauacucuuucagguccuguucgguuguuuuccaugggagacccaacacuauu (SEQ ID NO: 162) arRNA₁₁₁-CGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGagcaucgcgagcaggcFIG. 17G Reporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 163) arRNA₁₁₁-GGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugGagcaucgcgagcaggcReporter-3 (UAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 164) arRNA₁₁₁-UGA-gaugggcaccacccoggugaacagcuccucgcccuugcucacuggcagagcccuuGagcaucgcgagcaggcReporter-3 (UAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 165) arRNA₁₁₁-AGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaGagcaucgcgagcaggcReporter-3 (UAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 166) arRNA₁₁₁-CGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGggcaucgcgagcaggcReporter-3 (CAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 167) arRNA₁₁₁-GGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugGggcaucgcgagcaggcReporter-3 (CAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 168) arRNA₁₁₁-UGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuGggcaucgcgagcaggcReporter-3 (CAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 169) arRNA₁₁₁-AGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaGggcaucgcgagcaggcReporter-3 (CAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 170) arRNA₁₁₁-CGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 171) arRNA₁₁₁-GGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugGugcaucgcgagcaggcReporter-3 (AAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 172) arRNA₁₁₁-AGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaGugcaucgcgagcaggcReporter-3 (AAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 173) arRNA₁₁₁-UGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuGugcaucgcgagcaggcReporter-3 (AAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 174) arRNA₁₁₁-CGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGcgcaucgcgagcaggcReporter-3 (GAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 175) arRNA₁₁₁-GGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccugGcgcaucgcgagcaggcReporter-3 (GAC) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 176) arRNA₁₁₁-UGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuuGcgcaucgcgagcaggcReporter-3 (GAA) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 177) arRNA₁₁₁-AGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuaGcgcaucgcgagcaggcReporter-3 (GAU) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 178) arRNA₁₁₁-CGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGagcaucgcgagcaggcFIG. 17H Reporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 179) arRNA₁₁₁-GGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuGGagcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 180) arRNA₁₁₁-UGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuUGagcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 181) arRNA₁₁₁-AGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuAGagcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 182) arRNA₁₁₁-CGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGUgcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 183) arRNA₁₁₁-CGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGGgcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 184) arRNA₁₁₁-CGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGCgcaucgcgagcaggcReporter-3 (UAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 185) arRNA₁₁₁-CGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 186) arRNA₁₁₁-GGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuGGugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 187) arRNA₁₁₁-UGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuUGugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 188) arRNA₁₁₁-AGU-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccuAGugcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 189) arRNA₁₁₁-CGA-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGAgcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 190) arRNA₁₁₁-CGC-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGCgcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 191) arRNA₁₁₁-CGG-gaugggcaccaccccggugaacagcuccucgcccuugcucacuggcagagcccucGGgcaucgcgagcaggcReporter-3 (AAG) gcugccuccuccgcccugcagcuuguacagcucguccau(SEQ ID NO: 192) arRNA₁₁₁-KRAS-AG6gauucugaauuagcuguaucgucaaggcacucgugccgacgccaccagcuccaacCaccacaaguggagaguFIG. 17I cagucauuuucagcaggccucucucccgcaccugggagc (SEQ ID NO: 193)arRNA₁₁₁-KRAS-AG9gauucugaanuagcuggaucgucaaggcacucgggccgacgccaccagcuccaacCaccacaaguggagagucagucauuuucagcaggccucucucccgcaccggggagc (SEQ ID NO: 194) arRNA₁₁₁-TP53gggagcagccucuggcauucugggagcuucaucuggaccugggucuucagugaacCauuguucaauaucgucFIG. 23 cggggacagcaucaaaucauccauugcuugggacggcaa (SEQ ID NO: 195)arRNA₁₁₁-TP53-AG1gggagcagccucuggcauucugggagcuucaucuggaccugggucuucagugaacCauuguucaagaucguccggggacagcaucaaaucauccauugcuugggacggcaa (SEQ ID NO: 196)arRNA₁₁₁-TP53-AG4gggagcagccucuggcagucggggagcuucaucuggaccugggucuucagugaacCauuguucaagaucguccggggacagcaucaaaucauccagugcuugggacggcaa (SEQ ID NO: 197) arRNA₁₁₁-COL3A1cauauuacagaauaccuugauagcauccaauuugcauccuugguuagggucaaccCaguauucuccacucuuFIG. 26 gaguucaggauggcagaauuucaggucucugcaguuucu (SEQ ID NO: 198)arRNA₁₁₁-BMPR2gugaagauaagccaguccucuaguaacagaaugagcaagacggcaagagcuuaccCagucacuuguguggagacuuaaauacuugcauaaagauccauugggauaguacuc (SEQ ID NO: 199) arRNA₁₁₁-AHI1gugaacgucaaacugucggaccaauauggcagaaucuucucucaucucaacuuucCauauccguaucauggaaucauagcauccuguaacuacuagcucucuuacagcugg (SEQ ID NO: 200) arRNA₁₁₁-FANCCgccaaugaucucgugaguuaucucagcagugugagccaucagggugaugacauccCaggcgaucguguggcc(Site 2) uccaggagcccagagcaggaaguugaggagaaggugccu (SEQ ID NO: 201)arRNA₁₁₁-MYBPC3caagacggugaaccacuccauggucuucuugucggcuuucugcacuguguaccccCagagcuccguguugccgacauccugggguggcuuccacuccagagccacauuaag (SEQ ID NO: 202) arRNA₁₁₁-IL2RGaggauucucuuuugaaguauugcucccccaguggauuggguggcuccauucacucCaaugcugagcacuuccacagaguggguuaaagcggcuccgaacacgaaacgugua (SEQ ID NO: 203)arRNA₁₁₁-IDUA-V1gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggccCagagcugcuccucaucFIG. 29 cagcagcgccagcagccccauggccgugagcaccggcuu (SEQ ID NO: 204)arRNA₁₁₁-IDUA-V2gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggccCagagcugcuccucaucugcggggcgggggggggccgucgccgcguggggucguug (SEQ ID NO: 205)

It turned out that this linear guide RNA induced strong EGFP expressionin close to 40% of total cells harboring the Reporter-1 (FIG. 11B,upper). Because endogenous ADAR proteins could edit double-stranded RNA(dsRNA) substrates¹², we reasoned that the long guide RNAs could annealwith the target transcripts to form dsRNA substrates that in turnrecruit endogenous ADAR proteins for targeted editing. We thusdesignated such guide RNA as arRNA (ADAR-recruiting RNA).

To verify if endogenous ADAR proteins are indeed responsible for aboveobservation, we set out to examine the arRNA-mediated RNA editing inADAR-deficient cells. Since ADAR2 mRNA was barely detectable in HEK293Tcells (FIG. 12A), we generated HEK293T ADAR1^(−/−) cells, rendering thiscell line deficient in both ADAR1 and ADAR2 (FIG. 11C, d). Indeed, thedepletion of ADAR1 abrogated arRNA₇₀-induced EGFP signals (FIG. 11B,lower). Moreover, exogenous expression of ADAR1^(p110), ADAR1^(p150) orADAR2 in HEK293T ADAR1^(−/−) cells (FIG. 11C, d) successfully rescuedthe loss of EGFP induction by arRNA₇₀ (FIG. 11E, FIG. 12B),demonstrating that arRNA-induced EGFP reporter expression solelydepended on native ADAR1, whose activity could be reconstituted by itseither isoforms (p110 and p150) or ADAR2. Sanger sequencing analysis onthe arRNA₇₀-targeting region showed an A/G overlapping peak at thepredicted adenosine site within UAC; indicating a significant A to I (G)conversion (FIG. 11F). The next-generation sequencing (NGS) furtherconfirmed that the A to I conversion rate was about 13% of totalreporter transcripts (FIG. 11G). The quantitative PCR analysis showedthat arRNA₇₀ did not reduce the expression of targeted transcripts (FIG.13), ruling out the possible RNAi effect of the arRNA. Collectively, ourdata demonstrated that the arRNA is capable of generating significantlevel of editing on the targeted transcripts through the engineered A-Cmismatch.

Example 7. LEAPER Enables RNA Editing in Multiple Cell Lines

Because the expression of endogenous ADAR proteins is a prerequisite forLEAPER-mediated RNA editing, we tested the performance of LEAPER in apanel of cell lines originated from distinct tissues, including HT29,A549, HepG2, RD, SF268, SW13 and HeLa. We first examined the endogenousexpression of all three kinds of ADAR proteins using Western blottinganalyses. ADAR1 was highly expressed in all tested cell lines, and itsidentity in the Western blots was confirmed by the negative control,HEK293T ADAR1^(−/−) line (FIG. 14A, b). ADAR3 was detected only in HepG2and HeLa cells (FIG. 14A, b). ADAR2 was non-detectable in any cells, aresult that was not due to the failure of Western blotting because ADAR2protein could be detected from ADAR2-overexpressing HEK293T cells (FIG.14A, b). These findings are in consistent with previous reports thatADAR1 is ubiquitously expressed, while the expressions of ADAR2 andADAR3 are restricted to certain tissues¹¹.

We then set out to test the editing efficiencies of a re-designed 71-ntarRNA (arRNA₇₁) targeting the Reporter-1 (FIG. 15A and Sequences ofarRNAs and control RNAs used in this study listed above) in these celllines.

LEAPER worked in all tested cells for this arRNA₇₁, albeit with varyingefficiencies (FIG. 14C). These results were in agreement with the priorreport that the ADAR1/2 protein levels correlate with the RNA editingyield⁴², with the exception of HepG2 and HeLa cells. The suboptimalcorrelations of editing efficiencies with ADAR1 levels were likely dueto the abundant ADAR3 expressions in these two lines (FIG. 14A, b)because it has been reported that ADAR3 plays an inhibitory role in RNAediting. Importantly, LEAPER also worked in three different cell linesof mouse origin (NIH3T3, Mouse Embryonic Fibroblast (MEF) and B16) (FIG.14D), paving the way for testing its therapeutics potential throughanimal and disease models. Collectively, we conclude that LEAPER is aversatile tool for wide-spectrum of cell types, and for differentorganisms.

Example 8. Characterization and Optimization of LEAPER

To better characterize and optimize LEAPER, we investigated the choicesof nucleotide opposite to the adenosine within the UAG triplet of thetargeted transcript. In HEK293T cells, Reporter-1-targeting arRNA₇₁showed variable editing efficiencies with a changed triplet (5′-CNA, Ndenotes one of A/U/C/G) opposite to the targeted UAG (Sequences ofarRNAs and control RNAs used in this study listed above). A-C mismatchresulted in the highest editing efficiency, and the A-G mismatch yieldedthe least but evident edits (FIG. 16A). We then investigated thepreference of nucleotides flanking the A-C mismatch in arRNA. We testedall 16 combinations of 5′ and 3′ neighbor sites surrounding the cytidine(5′-N¹CN²) (Sequences of arRNAs and control RNAs used in this studylisted above), and found that the 3′ neighboring adenosine was requiredfor the efficient editing, while adenosine is the least favorablenucleotide at the 5′ site (FIG. 16B, c). We thus concluded that CCAmotif on the arRNA confers the highest editing efficiency targeting theUAG site. It is worthwhile to note that the 3′ neighboring guanosine(5′-N¹CG) in arRNA showed a dramatic inhibitory effect (FIG. 16B, c).

Length of RNA appeared relevant to arRNA efficiency in directing theediting on the targeted transcripts (FIG. 10C), consistent with aprevious report⁴². To fully understand this effect, we tested arRNAswith variable lengths targeting two different reportertranscripts—Reporter-1 and Reporter-2 (FIG. 15A, b). For either reportertargeting, arRNAs of 10 different sizes were designed and tested,ranging from 31-nt to 211-nt, with CCA triplet (for UAG targeting) rightin the middle (Sequences of arRNAs and control RNAs used in this studylisted above). Based on the reporter EGFP activities, the length ofarRNA correlated positively with the editing efficiency, for bothreporters, peaking at 111- to 191-nt (FIG. 16D). Although one arRNA₅₁appeared working, 71-nt was the minimal length for arRNA to work forboth reporters (FIG. 16D).

Next, we investigated the effect of the A-C mismatch position within anarRNA on editing efficiency. We fixed the lengths of all arRNAs fortesting to 71-nt, and slided the UAG-targeting ACC triplet from 5′ to 3′within arRNAs (Sequences of arRNAs and control RNAs used in this studylisted above). It turned out that placing the A-C mismatch in the middleregion resulted in high editing yield, and arRNAs with the mismatchsites close to the 3′ end outperformed those close to the 5′ end in bothreporters (FIG. 16E). For convenience, we placed the A-C mismatch at thecenter of arRNAs for all of our subsequent studies.

We also tested the targeting flexibility of LEAPER and tried todetermine whether UAG on target is the only motif subjected to RNAediting. For all 16 triplet combinations (5′-N¹AN²) on Reporter-3 (FIG.15C), we used the corresponding arRNAs with the fixed lengths (111-nt)and ensured the perfect sequencing match for arRNA and the reporterexcept for the editing site (A-C mismatch) (FIG. 16F and Sequences ofarRNAs and control RNAs used in this study listed above). NGS resultsshowed that all N¹AN² motifs could be edited. The UAN² and GAN² are themost and the least preferable motifs, respectively (FIG. 16F, g).Collectively, the nearest neighbor preference of the target adenosine is5′ U>C≈A>G and 3′ G>C>A≈U (FIG. 16G).

Example 9. Editing Endogenous Transcripts Using LEAPER

Next, we examined if LEAPER could enable effective editing on endogenoustranscripts. Using arRNAs of different lengths, we targeted the UAGmotifs in the transcripts of PPIB, KRAS and SMAD4 genes, and an UACmotif in FANCC gene transcript (FIG. 17A, Sequences of arRNAs andcontrol RNAs used in this study listed above). Encouragingly, targetedadenosine sites in all four transcripts were edited by theircorresponding arRNAs with all four sizes, albeit with variableefficiencies according to NGS results (FIG. 17B). In consistent with ourprior observation, longer arRNAs tended to yield higher editing rates.Of note, the 151-nt arRNA^(PPIB) edited ˜50% of total transcripts ofPPIB gene (FIG. 17B). No arRNAs showed RNAi effects on their targetedtranscripts (FIG. 18A) or ultimate protein level (e.g. KRAS, FIG. 18B).Besides, LEAPER is able to achieve desirable editing rate on non-UANsites (FIG. 17C and Sequences of arRNAs and control RNAs used in thisstudy listed above), showing the flexibility of LEAPER on editingendogenous transcripts. To further explore the power of LEAPER, wetested whether it could simultaneously target multiple sites. Weobserved multiplex editing of both TARDBP and FANCC transcripts byco-expression of two arRNAs (Sequences of arRNAs and control RNAs usedin this study listed above), with the efficiency even higher than thosewith individual arRNAs (FIG. 17D), indicating that LEAPER is well suitedfor editing multiple targets in parallel.

It is noteworthy that ADAR1/2 tend to promiscuously deaminate multipleadenosines in an RNA duplex⁴⁴ and the A-C mismatch is not the only motifto guide the A-to-I switch (FIG. 16A). It is therefore reasonable toassume that all adenosines on target transcripts within the arRNAcoverages are subjected to variable levels of editing, major sources ofunwanted modifications. The longer the arRNA, the higher the possibilityof such off-targets. We therefore examined all adenosine sites withinthe arRNA covering regions in these targeted transcripts. For PPIBtranscripts, very little off-target editing was observed throughout thesequencing window for variable sizes of arRNAs (FIG. 17E, f). However,in the cases of targeting KRAS, SMAD4 and FANCC genes, multipleoff-target edits were detected (FIG. 19A-0. For KRAS in particular, 11out of 30 adenosines underwent substantial A to I conversions in thesequencing window of arRNA₁₁₁ (FIG. 19A, b).

We next attempted to develop strategies to minimize such unwantedoff-target effects. Because an A-G mismatch suppressed editing for UAGtargeting (FIG. 16A), we postulated that pairing a guanosine with anon-targeting adenosine might reduce undesirable editing. We then testedthe effect of the A-G mismatch on adenosine in all possible tripletcombinations (5′-N₁AN²) as in Reporter-3 (FIG. 15C and Sequences ofarRNAs and control RNAs used in this study listed above). A-G mismatchindeed decreased the editing on adenosine in all tested targets, exceptfor UAG or AAG targeting (˜2%) (FIG. 17G), in comparison with A-Cmismatch (FIG. 16F). To further reduce editing rates at unwanted sites,we went on testing the effect of two consecutive mismatches. It turnedout that the additional mismatch at the 3′ end nucleotide of the tripletopposite to either UAG or AAG, abolished its corresponding adenosineediting (FIG. 17H and Sequences of arRNAs and control RNAs used in thisstudy listed above). In light of these findings, we attempted to applythis rule to reduce off-targets in KRAS transcripts (FIG. 19A). We firstdesigned an arRNA (arRNA₁₁₁-AG6) that created A-G mismatches on all“editing-prone” motifs covered by arRNA₁₁₁ (FIG. 17I, FIG. 19A andSequences of arRNAs and control RNAs used in this study listed above),including AAU (the 61^(st)), UAU (the 63^(rd)), UAA (the 65^(th)), AAA(the 66^(th)), UAG (the 94^(th)) and AAG (the 99^(th)). ThisarRNA₁₁₁-AG6 eliminated most of the off-target editing, while maintainedan on-target editing rate of ˜5%. In consistent with the findings inFIG. 17C the single A-G mismatch could not completely minimize editingin AAG motif (99^(th)) (FIG. 17I and FIG. 19A). We then added moremismatches on arRNA₁₁₁-AG6, including a dual mismatch (5′-CGG oppositeto the targeted motif 5′-AAG), plus three additional A-G mismatches tomitigate editing on the 27^(th), 98^(th) and the 115^(th) adenosines(arRNA₁₁₁-AG9) (Sequences of arRNAs and control RNAs used in this studylisted above). Consequently, we achieved a much improved specificity forediting, without additional loss of editing rate on the targeted site(A76) (FIG. 17I). In summary, engineered LEAPER incorporating additionalrules enables efficient and more precise RNA editing on endogenoustranscripts.

Example 10. RNA Editing Specificity of LEAPER

In addition to the possible off-target effects within the arRNA-covereddsRNA region, we were also concerned about the potential off-targeteffects on other transcripts through partial base pairing of arRNA. Wethen performed a transcriptome-wide RNA-sequencing analysis to evaluatethe global off-target effects of LEAPER. Cells were transfected with aCtrl RNA₁₅₁ or a PPIB-specific arRNA(arRNA₁₅₁-PPIB) expressing plasmidsbefore subjected to RNA-seq analysis. We identified six potentialoff-targets in the Ctrl RNA₁₅₁ group (FIG. 20A) and five in thearRNA₁₅₁-PPIB group (FIG. 20B), and the PPIB on-target rate based on NGSanalysis was ˜37% (FIG. 20B). Further analysis revealed that all sites,except for the two sites from EIF2AK2 transcripts, were located ineither SINE (Alu) or LINE regions (FIG. 20A, b), both are prone toADAR-mediated editing⁴⁵, suggesting that these off-targets may not bederived from pairing between the target transcripts and the arRNA orcontrol RNA. Of note, two off-targeting transcripts, WDR73 and SMYD4,appeared in both groups, suggesting they are unlikely sequence-dependentRNA editing. Indeed, minimum free energy analysis suggested that allthese possible off-target transcripts failed to form a stable duplexwith either Ctrl RNA₁₅₁ or arRNA₁₅₁-PPIB (FIG. 20C). To further test ifarRNA generates sequence-dependent off-targets, we selected potentialoff-target sites by comparing sequence similarity using NCBI BLAST forboth arRNA₁₅₁-PPIB and arRNA₁₁₁-FANCC. TRAPPC12 transcripts forarRNA₁₅₁-PPIB and three sites in the ST3GAL1, OSTM1-AS1 and EHD2transcripts for arRNA₁₁₁-FANCC were top candidates (FIG. 20D and FIG.21A). NGS analysis revealed that no editing could be detected in any ofthese predicted off-target sites (FIG. 20D and FIG. 21B). These resultsindicate that LEAPER empowers efficient editing at the targeted site,while maintaining transcriptome-wide specificity without detectablesequence-dependent off-target edits.

Example 11. Safety Assessment of LEAPER in Mammalian Cells

Because arRNAs rely on endogenous ADAR proteins for editing on targettranscripts, we wondered if the addition of exogenous arRNAs affectsnative RNA editing events by occupying too much of ADAR1 or ADAR2proteins. Therefore, we analyzed the A-to-I RNA editing sites shared bymock group and arRNA₁₅₁-PPIB group from the transcriptome-wideRNA-sequencing results, and the comparison between the mock group andCtrl RNA₁₅₁ group was also analyzed. Neither Ctrl RNA₁₅₁ group norarRNA₁₅₁-PPIB group showed a significant difference compared to the mockgroup (FIG. 22A, B), indicating that LEAPER had little impact on thenormal function of endogenous ADAR1 to catalyze the native A-to-Iediting events.

Meanwhile, we performed differential gene expression analysis usingRNA-seq data to verify whether arRNA affects global gene expression. Wefound that neither Ctrl RNA₁₅₁ nor arRNA₁₅₁-PPIB affected the globalgene expression in comparison with the mock group (FIG. 22C, D). Inconsistent with our prior observation (FIG. 18A), arRNAs did not showany RNAi effect on the expression of PPIB (FIG. 22C, D).

Considering that the arRNA forms RNA duplex with the target transcriptand that RNA duplex might elicit innate immune response, we investigatedif the introduction of arRNA has such an effect. To test this, weselected arRNAs targeting four gene transcripts that had been proveneffective. We did not observe any mRNA induction of interferon-β (IFN-β)(FIG. 22E) or interleukin-6 (IL-6) (FIG. 22F), which are two hallmarksof innate immune activation. As a positive control, a synthetic analogof double-stranded RNA-poly(LC) induced strong IFN-β and IL-6 expression(FIG. 22E, f). LEAPER does not seem to induce immunogenicity in targetcells, a feature important for safe therapeutics.

Example 12. Recovery of Transcriptional Regulatory Activity of p53 byLEAPER

Now that we have established a novel method for RNA editing without thenecessity of introducing foreign proteins, we attempted to demonstrateits therapeutic utility. We first targeted the tumor suppressor geneTP53, which is known to play a vital role in the maintenance of cellularhomeostasis, but undergo frequent mutations in >50% of human cancers⁴⁶.The c.158G>A mutation in TP53 is a clinically-relevant nonsense mutation(Trp53Ter), resulting in a non-functional truncated protein⁴⁶. Wedesigned one arRNA₁₁₁ and two alternative arRNAs (arRNA₁₁₁-AG1 andarRNA₁₁₁-AG4) (Sequences of arRNAs and control RNAs used in this studylisted above), all targeting TP53^(W53X) transcripts (FIG. 23A), withthe latter two being designed to minimize potential off-targets. Wegenerated HEK293T TP53^(−/−) cell line to eliminate the effects ofnative p53 protein. All three forms of TP53^(W53X)-targeting arRNAsconverted ˜25-35% of TP53^(W53X) transcripts on the mutated adenosinesite (FIG. 23B), with variable reductions of unwanted edits forarRNA₁₁₁-AG1 and arRNA₁₁₁-AG4 (FIG. 24). Western blot showed thatarRNA₁₁₁, arRNA₁₁₁-AG1 and arRNA₁₁₁-AG4 could all rescue the productionof full-length p53 protein based on the TP53^(W53X) transcripts inHEK293T TP53^(−/−) cells, while the Ctrl RNA₁₁₁ could not (FIG. 23C).

To verify whether the repaired p53 proteins are fully functional, wetested the transcriptional regulatory activity of p53 with ap53-luciferase cis-reporting system^(47, 48). All three versions ofarRNAs could restore p53 activity, and the optimized versionarRNA₁₁₁-AG1 performed the best (FIG. 23D). In conclusion, wedemonstrated that LEAPER is capable of repairing the cancer-relevantpre-mature stop codon of TP53 and restoring its function.

Example 13. Corrections of Pathogenic Mutations by LEAPER

We next investigated whether LEAPER could be used to correct morepathogenic mutations Aiming at clinically relevant mutations from sixpathogenic genes, COL3A1 of Ehlers-Danlos syndrome, BMPR2 of Primarypulmonary hypertension, AHI1 of Joubert syndrome, FANCC of Fanconianemia, MYBPC3 of Primary familial hypertrophic cardiomyopathy and IL2RGof X-linked severe combined immunodeficiency, we designed 111-nt arRNAsfor each of these genes carrying corresponding pathogenic G>A mutations(FIG. 25 and Sequences of arRNAs and control RNAs used in this studylisted above, and the disease-relevant cDNAs used in this study areshown in Table 4).

TABLE 4 Disease-related cDNAs used in this study Candidate DiseaseMutant Adenosine NM_000090.3 (COL3A1) Ehlers-Danlos syndrome, type 4c.3833G > A (p.Trp1278Ter) NM_001204.6 (BMPR2) Primary pulmonaryhypertension c.893G > A (p.Trp298Ter) NM_017651.4 (AHI1) Joubertsyndrome 3 c.2174G > A (p.Trp725Ter) NM_000136.2 (FANCC) Fanconi anemia,complementation group C c.1517G > A (p.Trp506Ter) NM_000256.3 (MYBPC3)Primary familial hypertrophic cardiomyopathy c.3293G > A (p.Trp1098Ter)NM_000206.2 (IL2RG) X-linked severe combined immunodeficiency c.710G > A(p.Trp237Ter)

By co-expressing arRNA/cDNA pairs in HEK293T cells, we identifiedsignificant amounts of target transcripts with A>G corrections in alltests (FIG. 24). Because G>A mutations account for nearly half of knowndisease-causing point mutations in humans^(10, 49), the A>G conversionby LEAPER may offer immense opportunities for therapeutics.

Example 14. RNA Editing in Multiple Human Primary Cells by LEAPER

To further explore the clinical utility of LEAPER, we set out to testthe method in multiple human primary cells. First, we tested LEAPER inhuman primary pulmonary fibroblasts and human primary bronchialepithelial cells with 151-nt arRNA (Sequences of arRNAs and control RNAsused in this study listed above) to edit the Reporter-1 (FIG. 15A).35-45% of EGFP positive cells could be obtained by LEAPER in both humanprimary cells (FIG. 27A). We then tested LEAPER in editing endogenousgene PPIB in these two primary cells and human primary T cells, andfound that arRNA₁₅₁-PPIB could achieve >40%, >80% and >30% of editingrates in human primary pulmonary fibroblasts, primary bronchialepithelial cells (FIG. 27B) and primary T cells (FIG. 27C),respectively. The high editing efficiency of LEAPER in human primarycells is particularly encouraging for its potential application intherapeutics.

Example 15. Efficient Editing by Lentiviral Expression and ChemicalSynthesis of arRNAs

We then investigated if LEAPER could be delivered by moreclinically-relevant methods. We first tested the effect of arRNA throughlentivirus-based expression. Reporter-1-targeting arRNA₁₅₁ inducedstrong EGFP expression in more than 40% of total cells harboring theReporter-1 in HEK293T cells 2 days post infection (dpi). At 8 dpi, theEGFP ratio maintained at a comparable level of ˜38% (FIG. 28A andSequences of arRNAs and control RNAs used in this study listed above),suggesting that LEAPER could be tailored to therapeutics that requirecontinuous administration. For native gene editing, we deliveredPPIB-targeting arRNA₁₅₁ through lentiviral transduction in HEK293T cellsand observed over 6% of target editing at 6 dpi (FIG. 28B).

We next tested synthesized arRNA oligonucleotides and electroporationdelivery method for LEAPER. The 111-nt arRNA targeting PPIB transcriptsas well as Ctrl RNA were chemically synthesized with 2′-O-methylationand phosphorothioate linkage at the first three and last threenucleotides of arRNAs (FIG. 28C). After introduced into T cells throughelectroporation, arRNA₁₁₁-PPIB oligos achieved ˜20% of editing on PPIBtranscripts (FIG. 28D), indicating that LEAPER holds promise for thedevelopment of oligonucleotide drugs.

Example 16. Restoration of α-L-Iduronidase Activity in Hurler SyndromePatient-Derived Primary Fibroblast by LEAPER

Finally, we examined the potential of LEAPER in treating a monogenicdisease—Hurler syndrome, the most severe subtype ofMucopolysaccharidosis type I (MPS I) due to the deficiency ofα-L-iduronidase (IDUA), alysosomal metabolic enzymeresponsible for thedegradation of mucopolysaccharides⁵⁰. We chose a primary fibroblastGM06214 that was originally isolated from Hurler syndrome patient. TheGM06214 cells contain a homozygous TGG>TAG mutation in exon 9 of theIDUA gene, resulting in a Trp402Ter mutation in the protein. We designedtwo versions of arRNAs by synthesized RNA oligonucleotides with chemicalmodifications of 2′-O-methylations and internucleotidephosphorothiatelinkages in the first and last 3 nucleotides of thesequences, arRNA₁₁₁-IDUA-V1 and arRNA₁₁₁-IDUA-V2, targeting the maturemRNA and the pre-mRNA of IDUA, respectively (FIG. 29A and Sequences ofarRNAs and control RNAs used in this study listed above). Afterintroduction of arRNA₁₁₁-IDUA-V1 or arRNA₁₁₁-IDUA-V21 into GM06214 cellsvia electroporation, we measured the targeted RNA editing rates via NGSanalysis and the catalytic activity of α-L-iduronidase with4-MU-α-L-iduronidase substrate at different time points. BotharRNA₁₁₁-IDUA-V1 and arRNA₁₁₁-IDUA-V2 significantly restored the IDUAcatalytic activity in IDUA-deficient GM06214 cells progressively withtime after electroporation, and arRNA₁₁₁-IDUA-V2 performed much betterthan arRNA₁₁₁-IDUA-V1, while no α-L-iduronidase activity could bedetected in three control groups (FIG. 29B).

To further evaluate the extent to which the restored IDUA activity inGM06214 by LEAPER relieves the Hurler syndrome, we examined the IDUAactivity in GM01323 cells, another primary fibroblasts from patient withScheie syndrome, a much milder subtype of MPS I than Hurler syndrome dueto the remnant IDUA activity resulting from heterozygous genotype onIDUA gene. We found that the catalytic activity of IDUA in GM06214 cellsharboringarRNA₁₁₁-IDUA-V2 was higher than GM01323 cells 48 hr postelectroporation (FIG. 29B). Consistent with these results, NGS analysisindicated that arRNA₁₁₁-IDUA-V2 converted nearly 30% of A to I editing,a much higher rate than arRNA₁₁₁-IDUA-V1 (FIG. 29C). Further analysisrevealed that minimal unwanted edits were detected within the arRNAcovered regions of IDUA transcripts (FIG. 29D). Importantly, LEAPER didnot trigger immune responses in primary cells as we demonstrated that,unlike the RNA duplex poly(I:C) serving as a positive control, neitherarRNA₁₁₁-IDUA-V1 nor arRNA₁₁₁-IDUA-V2 induced expressions of a panel ofgenes involved in type-I interferon and pro-inflammatory responses (FIG.29E). These results showed the therapeutic potential of LEAPER intargeting certain monogenetic diseases.

Example 17. Detection of GM06214 Mutant Genotype

GM06214 cells was cultured in a fibroblast culture medium (ScienCell, FMmedium, Cat. No. 2301) containing 15% serum and 1% fibroblast growthadditive (ScienCell, GFS, Cat. No. 2301), in an incubator of 37° C. and5% CO₂, for 2-3 days. When cells are 90% confluent, they are digestedwith 0.25% trypsin, then the digestion is terminated by fibroblastculture medium containing 15% serum. DNA extraction was performed usinga TianGene® (TIANGEN Biotech (Beijing) Co., Ltd.) cell DNA extractionkit (Cat. No. DP304-03) according to the operating instructions.

Primers for sequences upstream and downstream of the IDUA mutation sitewas designed using NCBI-Primer blast (website:https://www.ncbi.nlm.nih.gov/tools/primer-blast/). SEQ ID NO:304:CGCTTCCAGGTCAACAACAC (forward primer hIDUA-F1); SEQ ID NO 305:CTCGCGTAGATCAGCACCG (reverse primer hIDUA-R1). A PCR was performed, andthe PCR products were subjected to Sanger sequencing. As shown in FIG.34, the mutation of the cells was confirmed to be a G to A mutationwhich results in the disease.

Example 18. Test of GM06214 Cell Transfection Conditions

GM06214 cells were digest when the GM06214 at 90% confluency, and werecounted after the terminating of digestion. For electrotransfection, 6million cells were resuspend with 400 ul of pre-mixedelectrotransfection solution (Lonza, Cat. No. V4XP-3024), and added with20 ug of GFP plasmid (Lonza, Cat. No. V4XP-3024). After mixing, 20ul ofthe suspension is taken as an electrotrasfection system for the test ofeach of the 8 conditions, comprising 7 test electrotransfectionconditions (see FIG. 35) and one negativecontrol, using a LonzaNucleofector™ instrument. The test of each condition is duplicated.After electrotransfection, the cells are rapidly transferred into 2 mlfibroblast culture medium (ScienCell, FM medium, Cat. No. 2301)containing 15% serum. Cells of each condition were plated into 2 wells(6 well culture plates) and cultured in an incubator of 5% CO2 and 37°C. 24 hours after electrotransfection, cells in one of the 2 wells ofeach electrotransfection condition were digested, and the proportion ofGFP-positive cells was measured by flow cytometry. 48 hours afterelectrotransfection, the cells in the other well of the 2 wells of eachelectrotransfection condition are digested, and the proportion ofGFP-positive cells was measured by flow cytometry. The optimalelectrotransfection conditions for the cells are CA-137 conditions, asshown in FIG. 35.

Example 19. Detection of IDUA Enzyme Activity and A to G Mutation Rate

The oligo dRNAs are designed and synthesized for targeting the sequencewith the mutation site of the pre-mRNA and mature RNA transcripted fromIDUA gene. The sequence of the dRNAs are shown as follows. All the dRNAsequences were modified in CM0 pattern (2′-O-methylations were in thefirst and last 3 nucleotides of the sequences and the first and last 3internucleotide linkages in the sequences were phosphorothiated).

SEQ ID NO 204: gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucauccagcagcgccagcagccccauggccgugag caccggcuu(Pre-55nt-c-55nt); SEQ ID NO 205:gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccgucgccgcgug gggucguug(m-55nt-c-55nt); SEQ ID NO 341:uaccgcuacagccacgcugauuucagcuauaccugcccgguauaaagggacguucacaccggauguucuccgcggggauaucgcgauauucaggauuaaaag aagugc(Random-111nt).

Wherein the base corresponding to the mutated base in the synthesizeddRNA is a C, which forms an A-C mismatch with the mutated base whenbinding. The length of the synthesized dRNA is preferably 111 nt. Thecells were electrotransfected using the optimal electrotransfectioncondition obtained in Example 2. 48 hours after electrotransfection, thecells were collected for enzyme activity determination and A to Gmutation rate detection.

Determination of A to G Mutation Rate:

The designed dRNA was dissolved to the required concentration inRNase-free water (TransGene Biotech, Cat. No. GI201-01) and stored at−80° C. Cells were digested when the GM06214 cells grow to about 90%confluence and counted after the terminating of the digestion. 1 millioncells and 200 pmol of dRNA were mixed and diluted to 100 ul, and thenelectrotransfected under the condition of CA-137. 48 hours afterelectrotransfection, cells were counted and their viability wasmeasured. The cells were transferred to a RNase-free centrifuge tube andcentrifuged. The supernatant was discarded. RNA was extracted using aQIAGEN RNA extraction kit (QIAGEN, Cat. No. 74134). According to theinstructions, 0.35 ml of Buffer RLT Plus was mixed with 5×10⁵ cells (ifthe RNA is directly extracted from frozen cells, it is recommended thatcells be washed with PBS once) by pipetting. The cell lysate wastransferred to the gDNA Eliminator spin column and centrifuged at 8000 gfor 30 s. The column was discarded and the liquid was remained. The samevolume of 70% ethanol as the liquid was added. Immediatedly aftermixing, the mixture was transferred to the RNeasyMinElute spin columnand centrifuged at ≥8000 g for 15 sand the waste liquid was discarded.700 μl of Buffer RW1 was added to the RNeasyMinElute spin column andcentrifuged at 8000 g for 15 s. Waste solution was discarded and 500 μlof Buffer RPE was added, and then the RNeasyMinElute spin column wascentrifuge at ≥8000 g for 15 s. Waste solution was discarded and 500 μlof 80% ethanol was added, and then the RNeasyMinElute spin column wascentrifuged at ≥8000 g for 2 minutes. Waste solution was discarded. TheRNeasyMinElute spin column was placed into a new 2 ml collection columnand centrifuged with the lid at maximum speed for 5 minutes to dry thecolumn. The RNeasyMinElute spin column was placed into a new 1.5 mlcollection column and 14 μl of RNase-free water was added dropwise tothe center of the column membrane, then the columns are centrifuged atmaximum speed for 1 minute to elute the RNA.

The consentrition of the extracted RNA was determined by Nanodrop(Thermo, Nanodrop2000), and 1 ug of RNA was used for reversetranscription (Thermo, reverse transcriptase, Cat. No. 28025013). Thereverse transcription system was shown in Table 5-6. After incubation at65° C. for 5 minutes, the reverse transcription system was immediatelycooled in an ice bath. Incubation was continued at 37° C. for 50minutes. Reverse transcriptase was inactivated at 70° C. for 15 minutes.PCR was performed under the conditions shown in Table 7. After PCR, 2 ulof the PCR product was taken for agarose gel electrophoresis. Accordingto the results of the electrophoresis, the concentration of the PCRproduct and whether the band size is correct is determined. Afterpurification, the PCR products were used to preparing the library whichwas sent for next-generation sequencing.

TABLE 5 Reverse transcription system-1 Volume (ul) Total RNA(lug) XOligo dT  1 10 nM dNTP  1 RNase-Free Water 10-X Total volume 12 65° C.,5 min, and immediately transferring to the ice

TABLE 6 Reverse transcription system-2 Volume (ul) The product fromTable 5 12 ul 5X First-Strand Buffer 4 0.1M DTT 2 RNaseOUT ™ Recombinant1 Ribonuclease Inhibitor M-MLV 1 Total volume 20

TABLE 7 PCR conditions Steps Time Cycle 98° C.  2 min 1 cycle 98° C. 15s 28-35 cycle 63° C. 30 s 72° C. 15 s 72° C.  2 min 1 cycleEnzyme Activity Assay in this Example:

GM06214 cells were digested, centrifuged, and resuspended in 28 ul of1×PBS containing 0.1% Triton X-100 and lysed on ice for 30 minutes. Then25 ul of cell lysate was added to 25 ul of substrate containing 190 μm4-methylumbelliferyl-α-L-iduronidase (Cayman, 2A-19543-500, Dissolved in0.4 M sodium formate buffer containing 0.2% Triton X-100, pH 3.5) andincubated in the dark at 37° C. for 90 minutes. 200 ul 0.5M NaOH/Glycinesolution (Beijing Chemical Works, NAOH, Cat. No. AR500G; Solarbio,Glycine, Cat. No. G8200), pH 10.3, was added to inactivate the catalyticreaction. After centrifuging at 4° C. for 2 minutes, its supernatant wastransferred to a 96-well plate for the determination of fluorescencevalues using Infinite M200 instrument (TECAN). The wavelength of theexcitation light was 365 nm and 450 nm. The fluorescence represents theenzyme activity which in the figures is expressed as a multiple of theenzyme activity in GM01323.

As shown in FIG. 36, the results were that dRNA targetingpre-mRNAleading to significantly higher enzyme activity and A to Gmutation rate than those targeting mature-mRNA. Therefore, the dRNAsused in the following examples are targeted to pre-mRNA.

Example 20. Detection of Editing Efficiency in IDUA-Reporter Cell Lineafter Electrotransfection of Chemically Modified dRNA

As shown in FIG. 37A, a plasmid was constructed by inserting a sequencewith an IDUA mutation site flanked with about 100 bp on each side,respectively, between the sequences expressing mcherry and GFP proteinson the lentiviral plasmid. The constructed plasmids were packaged intoviruses used to infect 293T cells later. After integration into thegenome, IDUA-reporter monoclonal cells were selected. Because themonoclonal cells were affected by the TAG stop codon of the IDUAmutation site in the inserted sequence, they only expressed the mcherryprotein. When the cells are edited by dRNA, the GFP behind TAG which hasthen been mutated to TGG can express normally. Thus, the expression ofGFP was viewed as the editing efficiency of dRNA in cells. 4 preferabledRNAs with different lengths from 51 nt to 111 nt were designed, asshown in Table 8 below. All the dRNA sequences were modified in CM0pattern. Cells were electrotransfected with dRNAs of different lengthsunder the conditions of electrotransfection in Example 18. On each dayfrom the 1th day to the 7thday after the transfection, the editingefficiency was preliminarily evaluated by determining the ratio of GFPin the cells. As shown in FIG. 37B, the peak of editing efficiencyappeared on the second day (48 h). The sequence with the highest editingefficiency was 91 nt: 45-c-45 which is higher than that of 111 nt:55-c-55. Accordingly, it's not in all cases that the longer the dRNA,the higher the editing efficiency. Besides, the editing efficiency ofdRNAs of 51 nt was very low.

TABLE 8 111nt- SEQ ID NO: 140: randomuaccgcuacagccacgcugauuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucucugcuggggaauug cgcgauauucaggauuaaaagaagugc91nt- SEQ ID NO: 342: random uaauccugaauaucgcgcaauuccccagcagagaacaucgcggugugaacgucccuuuauaccgggcagguauagcugaaauca gcguggc 71nt- SEQ ID NO: 343:random uuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucucugcuggggaauugcgcgaua 51nt- SEQ ID NO 8: randomuuccccagcagagaacaucgcggugugaacgucccuuuauac cgggcaggu 55nt-c-SEQ ID NO: 205: 55nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggg gggggccgucgccgcguggggucguug45nt-c- SEQ ID NO: 344: 45nt gugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccguc gccgcgu 35nt-c-SEQ ID NO: 345: 35nt uguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggcc 25nt-c- SEQ ID NO: 346: 25ntggucccggccugcgacacuucggcccagagcugcuccucauc ugcggggcg

Example 21. Determination of the Intracellular IDUA Enzyme Activity andRNA Editing Efficiency in GM06214 Cells at Different Time Points afterTransfection with Chemically Modified dRNAs of Different Lengths

The conditions in Example 18 (see Table 7) for electrotransfecting dRNAsof different lengths into GM06214 cells and the methods in Example 19for determining enzyme activity and editing efficiency were used. On the2th, 4th, 6th, 8th, 10^(th), 12^(th) and 14^(th) after theelectrotransfection, the intracellular enzyme activity was tested. Andon the 2th and 4^(th) day, the efficiency of RNA editing in the cellswas tested. As shown in FIG. 38A, 91 nt: 45-c-45 led to the highestenzyme activity, and the IDUA enzyme activity had been maintained at ahigh level till the 6th day after electrotransfection. In FIG. 38B, dRNAof 91 nt and dRNA of 111 nt presented roughly the same editingefficiency. Again, the dRNA of 51 nt showed a low editing efficiency.

Example 22. Screening for Preferable Sequences of Chemically ModifieddRNAs

Through literature research, we believe electrotransfection is notsuitable for disease treatment in the future. Therefore, we turnedelectrotransfection to Lipofectamine RNAiMAX ((Invitrgen, Cat. No.13778-150)) for transfecting dRNA into cells. It turned out that theLipofectamine RNAiMAX has a higher transfection efficiency than that ofelectrotransfection. The sequence was first truncated on both termini atthe same time, and then one terminus of the sequence is fixed and theother terminus was truncated. In this way, 14 dRNAs and 4 randomsequences of equal length are obtained, as shown in Table 9 below. Allthe dRNA sequences were modified in CM0 pattern. As shown in FIG. 39,the IDUA enzyme activity (FIG. 39A, using the method described inExample 19) and RNA editing efficiency (FIG. 39B, using NGS) weredetermined 48 hours after transfection. The IDUA enzyme activities andRNA editing efficiencies led by 81 nt: 55-c-25 (SEQ ID NO 24) and 71 nt:55-c-15 (SEQ ID NO 25) turned out to be higher than that led by theother dRNAs. And RNA with a shorter 3′ terminus and a longer 5′ terminusalways had a higher efficiency. In addition, it seems that the editingefficiency of dRNA decreased dramatically when its length was reduced to61 nt or less, no matter how the 3′ or 5′ terminus changed.

TABLE 9 111nt- SEQ ID NO: 140: randomuaccgcuacagccacgcugauuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucucugcuggggaauug cgcgauauucaggauuaaaagaagugc91nt- SEQ ID NO: 342: random uaauccugaauaucgcgcaauuccccagcagagaacaucgcggugugaacgucccuuuauaccgggcagguauagcugaaauca gcguggc 71nt- SEQ ID NO: 343:random uuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucucugcuggggaauugcgcgaua 51nt- SEQ ID NO 8: randomuuccccagcagagaacaucgcggugugaacgucccuuuauac cgggcaggu 55nt-c-SEQ ID NO: 205: 55nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggg gggggccgucgccgcguggggucguug45nt-c- SEQ ID NO: 344: 45nt gugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccguc gccgcgu 35nt-c-SEQ ID NO 345: 35nt uguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggcc 25nt-c- SEQ ID NO: 346: 25ntggucccggccugcgacacuucggcccagagcugcuccucauc ugcggggcg 55nt-c-SEQ ID NO: 347: 45nt gacgcccaccguguggaugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggg gggggccgucgccgcgu 55nt-c-SEQ ID NO: 348: 35nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggg gggggcc 55nt-c-SEQ ID NO: 349: 25nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcg 55nt-c- SEQ ID NO: 350: 15ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcuccucau55nt-c- SEQ ID NO: 351: 5nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagc 45nt-c- SEQ ID NO: 352: 55ntgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccguc gccgcguggggucguug 35nt-c-SEQ ID NO: 353: 55nt uguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccgucgccgcguggg gucguug 25nt-c-SEQ ID NO: 354: 55nt ggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcgggggggggccgucgccgcguggggucguug 15nt-c- SEQ ID NO: 355: 55ntugcgacacuucggcccagagcugcuccucaucugcggggcgg gggggggccgucgccgcguggggucguug5nt-c- SEQ ID NO: 356: 55nt cggcccagagcugcuccucaucugcggggcgggggggggccgucgccgcguggggucguug

Example 23. Determination of the Optional Length of the 3′ Terminus ofChemically Modified dRNA

In Example 22, higher IDUA enzyme activity and editing efficiency weredetected in cells edited by dRNAs with 81 nt: 55-c-25 and 71 nt: 55-c-15sequences. In order to find out the shortest and optimal length of the3′ terminus, the sequence at 3′ terminus of was truncated from 25 nt (81nt: 55-c-25) to 5 nt (61 nt: 55-c-5), as shown in Table 10. All the dRNAsequences were modified in CM0 pattern. Two IDUA enzyme activity assayswere conducted on cells separately transfected with dRNAs from 81 nt:55-c-25 to 66 nt: 55-c-10 (FIG. 40A) and cells separately transfectedwith dRNAs from 72 nt: 55-c-16 to 61 nt: 55-c-5 (FIG. 40B). The dRNAswith the 3′ terminus lengths from 25 nt to 9 nt easily raised theenzymatic activity in GM06214 cells to more than 20 times of that inGM0123 cells. Accordingly, the optimal length of the 3′ terminus was 25nt-7 nt. Besides, compared to 45 nt-c-45 nt having equal length of 3′and 5′ termini, the dRNAs with shorter 3′ termini always had higherediting efficiency.

The IDUA enzyme activity assay used herein is described as below. Oneday before transfection, 3×10⁵ cells per well were plated in a 6-wellplate. Medium was refreshed on the day of transfection. 48 hrs aftertransfection using 20 nM Lipofectamine RNAiMAX reagent, GM06214 cellswere digested, centrifuged, and resuspended in 33 ul of 1×PBS containing0.1% Triton X-100 and lysed on ice for 30 minutes. Then the lysate wascentrifuged at 4° C. for 2 min. 25 ul of cell lysate was added to 25 ulof substrate containing 190 μm 4-methylumbelliferyl-α-L-iduronidase(Glycosynth, 44076) dissolved in 0.4 M sodium formate buffer containing0.2% Triton X-100 (pH 3.5) and incubated in the dark at 37° C. for 30minutes. 200 ul 0.5M NaOH/Glycine solution (Beijing Chemical Works,NAOH, Cat. No. AR500G; Solarbio, Glycine, Cat. No. G8200), pH 10.3, wasadded to inactivate the catalytic reaction. All of its supernatant wasdetected using Infinite M200 instrument (TECAN). The wavelength of theexcitation light was 365 nm and 450 nm. The enzyme activity is expressedas a multiple of the enzyme activity in GM01323.

TABLE 10 55nt-c- SEQ ID NO: 349: 25ntgacgcccaccgugagguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggcg 55nt-c- SEQ ID NO: 357: 24ntgacgcccaccgugagguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggggc 55nt-c- SEQ ID NO: 358: 23ntgacgcccaccgugagguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgggg 55nt-c- SEQ ID NO: 359: 22ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggg 55nt-c- SEQ ID NO: 360: 21ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgg 55nt-c- SEQ ID NO: 361: 20ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg 55nt-c- SEQ ID NO: 362: 19ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugc 55nt-c- SEQ ID NO: 363: 18ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucug 55nt-c- SEQ ID NO: 364: 17ntgacgcccaccguguggaugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucu 55nt-c- SEQ ID NO: 365: 16ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucauc 55nt-c- SEQ ID NO: 350: 15ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcuccucau55nt-c- SEQ ID NO: 366: 14nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccuca 55nt-c- SEQ ID NO: 367: 13ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcuccuc55nt-c- SEQ ID NO: 368: 12nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccu 55nt-c- SEQ ID NO: 369: 11ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcucc55nt-c- SEQ ID NO: 370: 10nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuc 55nt-c- SEQ ID NO: 371: 9ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcu55nt-c- SEQ ID NO: 372: 8nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugc 55nt-c- SEQ ID NO: 373: 7ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcug 55nt-c-SEQ ID NO: 374: 6nt gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcu 55nt-c- SEQ ID NO: 375: 5ntgacgcccaccguguggaugcuguccaggacggucccggccug cgacacuucggcccagagc random-SEQ ID NO: 376: 70nt uaccgcuacagccacgcugauuucagcuauaccugcccgguauaaagggacguucacaccgcgauguucu random- SEQ ID NO: 377: 67ntuaccgcuacagccacgcugauuucagcuauaccugcccggua uaaagggacguucacaccgcgaug

Example 24. Determination of the Optional Length of 5′ Terminus ofChemically Modified dRNA when the Length of its 3′ Terminus was Fixed

The truncation of 5′ terminus was separately conducted ond RNAs of twodifferent lengths: 76 nt: 55-c-20 and 71 nt: 55-c-15. With the fixedlength of 3′ terminus, their 5′ termini were gradually truncated, asshown in Table 11. All the dRNA sequences were modified in CM0 pattern.According to the result of IDUA enzyme activity assay, cells transfectedwith dRNAs with 5′ terminals between 55 nt and 45 nt had higher IDUAenzyme activity, as shown in FIG. 41. Lipofectamine RNAiMAX was used inthe transfection. In accordance with FIG. 39, when the length wasreduced to less than 61 nt, the editing efficiency of dRNAs, even thosewith unequal lengths of 3′ and 5′ termini, decreased dramatically.

TABLE 11 55nt-c- SEQ ID NO: 361: 20ntgacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg 50nt-c- SEQ ID NO: 378: 20ntccaccgugugguugcuguccaggacggucccggccugcgaca cuucggcccagagcugcuccucaucugcg45nt-c- SEQ ID NO: 379: 20nt gugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg 40nt-c- SEQ ID NO: 380: 20ntguugcuguccaggacggucccggccugcgacacuucggccca gagcugcuccucaucugcg 35nt-c-SEQ ID NO: 381: 20nt uguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg 55nt-c- SEQ ID NO: 350: 15ntgacgcccaccgugugguugcuguccaggacggucccggccug cgacacuucggcccagagcugcuccucau50nt-c- SEQ ID NO: 382: 15nt ccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucau 45nt-c- SEQ ID NO: 383: 15ntgugugguugcuguccaggacggucccggccugcgacacuucg gcccagagcugcuccucau 40nt-c-SEQ ID NO: 384: 15nt guugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucau 35nt-c- SEQ ID NO: 385: 15ntuguccaggacggucccggccugcgacacuucggcccagagcu gcuccucau

Example 27. Determination of the Relation Between the TargetingNucleotide Location and the Editing Efficiency of Chemically ModifieddRNAs to the IDUA Mutation Site

According to the data above, the editing efficiency of dRNA is relatedto the length and the location of the targeting nucleotide on the dRNA.Usually, the closer the targeting nucleotide is to the 5′ end, the lowerthe editing efficiency is. Thus, in this example, 3 groups of dRNAs of 3fixed lengths were designed. dRNAs in each group were designed bygradually moving the targeting nucleotide from the middle of thesequence toward the 5′ end. Structures that are not easy to synthesizeare avoided. Sequences are shown in Table. 12. All the dRNA sequenceswere modified in CM0 pattern. The dRNAs were transfected into GM06214cells using Lipofectamine RNAiMAX. 48 hrs later, the cells wereharvested and the enzyme activities were tested according to the methodsdescribed in Example 23. According to the data shown in FIG. 42, atleast when the total length of dRNA was fixed to 67 nt, 70 nt or 72 nt,the location change of the targeting nucleotide didn't seem to affectthe enzyme activity which represented the editing efficiency.

TABLE 12 Location Sequence Length of C No. Sequence 67nt 55nt-c-11ntSEQ ID NO:gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccsliding 369 54nt-c-12nt SEQ ID NO:acgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccu 38653nt-c-13nt SEQ ID NO:cgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccuc 38752nt-c-14nt SEQ ID NO:gcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccuca 38851nt-c-15nt SEQ ID NO:cccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucau 38950nt-c-16nt SEQ ID NO:ccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucauc 39049nt-c-17nt SEQ ID NO:caccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucu 39148nt-c-18nt SEQ ID NO:accgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucug 39247nt-c-19nt SEQ ID NO:ccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugc 39346nt-c-20nt SEQ ID NO:cgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg 39445nt-c-21nt SEQ ID NO:gugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgg 39544nt-c-22nt SEQ ID NO:ugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggg 39643nt-c-23nt SEQ ID NO:gugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugegggg 39770nt 55nt-c-14nt SEQ ID NO:gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucasilding 366 54nt-c-15nt SEQ ID NO:acgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucau398 53nt-c-16nt SEQ ID NO:cgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucauc399 52nt-c-17nt SEQ ID NO:gcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucu400 51nt-c-18nt SEQ ID NO:cccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucug401 50nt-c-19nt SEQ ID NO:ccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugc402 49nt-c-20nt SEQ ID NO:caccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg403 48nt-c-21nt SEQ ID NO:accgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgg404 47nt-c-22nt SEQ ID NO:ccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcggg405 46nt-c-23nt SEQ ID NO:cgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgggg406 72nt 55nt-c-16nt SEQ ID NO:gacgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucasliding 365 uc 54nt-c-17nt SEQ ID NO:acgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucau407 cu 53nt-c-18nt SEQ ID NO:cgcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucauc408 ug 52nt-c-19nt SEQ ID NO:gcccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucu409 gc 51nt-c-20nt SEQ ID NO:cccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucug410 cg 50nt-c-21nt SEQ ID NO:ccaccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugc411 gg 49nt-c-22nt SEQ ID NO:caccgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcg412 gg 48nt-c-23nt SEQ ID NO:accgugugguugcuguccaggacggucccggccugcgacacuucggcccagagcugcuccucaucugcgg413 gg

Example 28. Effect of Chemical Modification on Editing Efficiency ofdRNA

Chemical modifications of synthesized RNA increase RNA stability andreduce off-target potential. The relatively common chemicalmodifications of RNA are 2′-O-methylation (2′-O-Me) and phosphorothioatelinkage. The dRNAs with different combinations of lengths: 71 nt or 76nt and chemical modifications were shown in Table 13. GM06214 cells weretransfected with the different dRNAs using Lipofectamine RNAiMAX for theediting of intracellular IDUA. Cells were collected 48 hours aftertransfection, and IDUA enzyme activity were determined using the methodshown in Example 23. According to the results shown in FIG. 42A, all themodifications led to excellent enzyme activities, except for CM5 (the5th modification: all nucleotides, except for the targeting nucleotideand 5 nt on each side of it, were modified by2′-OMe). The modificationon the targeting nucleotide or the two nucleotides most adjacent to itdidn't reduce the editing efficiency.

The editing efficiency was further determined by counting the A to Gsubstitution rate. The method was described as below: A sequencecomprising the target adenosine in IDUA gene of GM06214 cells is CTAGwhich is mutated to CTGG after RNA editing using dRNAs. CTAG is therecognition site of restriction enzyme BfaI. Thus, a successful A to Gsubstitution doesn't result in a digestion by BfaI, while the wild typedoes. After editing, RNA of GM06214 cells were extracted and reversetranscribed into cDNA. PCR were conducted using the cDNA. Primers werehIDUA-62F: CCTTCCTGAGCTACCACCCG (SEQ ID NO: 415) and hIDUA-62R:CCAGGGCTCGAACTCGGTAG (SEQ ID NO: 416). After PCR, the product waspurified and incubated with BfaI (NEB, Cat. No. R0568L). The A to Gsubstitution rate, or the editing efficiency was determined usingagarose gel electrophoresis. The result was expressed as the percentageof the uncut sections (with A to G substitution) to the total nucleicacid in the PCR product, calculated using the gray values of the gelelectrophoresis image. The result was shown in FIG. 42B. It was similarto the result of enzyme activity assay in FIG. 42A.

TABLE 13 Name Length Modification pattern Sequence HIV2-76-CM1 55nt-c-CM1: Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 20ntModifications in CM0Um-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G- and all U: are withA-C-A-C-Um-Um-C-G-G-C-C-C-A-G-A-G-C-Um-G-C-Um-C-C- 2′-OMeUm-C-A-Um-C-Um*Gm*Cm*Gm (SEQ ID NO: 361) HIV2-76-CM2 55nt-c- CM2:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 20nt Modifications inUm-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G-CM1 and the targetingA-C-A-C-Um-Um-C-G-G-C-C-C-Am-G-A-G-C-Um-G-C-Um-C-C- triplet is CCAmUm-C-A-Um-C-Um*Gm*Cm*Gm (SEQ ID NO: 361) HIV2-76-CM3 55nt-c- CM3:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 20nt Modifications inUm-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G-CM1 and the targetingA-C-A-C-Um-Um-C-G-G-C-Cm-C-A-G-A-G-C-Um-G-C-Um-C-C- triplet is CmCAUm-C-A-Um-C-Um*Gm*Cm*Gm (SEQ ID NO: 361) HIV2-76-CM4 55nt-c- CM4:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 20nt Modifications inUm-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G-CM1 and the targeting A-C-A-C-Um-Um-C-G-G-C-C*C*A*G-A-G-C-Um-G-C-Um-C-C-triplet is C*C*A* Um-C-A-Um-C-Um*Gm*Cm*Gm (SEQ ID NO: 361) HIV2-76-CM555nt-c- CM5: Gm*Am*Cm*Gm-Cm-Cm-Cm-Am-Cm-Cm-Gm-Um-Gm-Um-Gm- 20ntModifications in CM1 Gm-Um-Um-Gm-Cm-Um-Gm-Um-Cm-Cm-Am-Gm-Gm-Am-Cm-and all nucleotides Gm-Gm-Um-Cm-Cm-Cm-Gm-Gm-Cm-Cm-Um-Gm-Cm-Gm-Am-with 2′-OMe, except Cm-Am-Cm-Um-Um-C-G-G-C-C-C-A-G-A-G-C-Um-Gm-Cm-Um-for the targeting Cm-Cm-Um-Cm-Am-Um-Cm-Um*Gm*Cm*Gm (SEQ ID NO: 361)nucleotide and 5nt on each side of it HIV2-76-CM6 55nt-c- CM6:Gm*Am*Cm*Gm*Cm*C-C-A-C-C-G-U-G-U-G-G-U-U-G-C-U-G- 20nt5 terminal bases atU-C-C-A-G-G-A-C-G-G-U-C-C-C-G-G-C-C-U-G-C-G-A-C-A-C-U-each terminus are withU-C-G-G-C-C-C-A-G-A-G-C-U-G-C-U-C-C-U-C-A-U*Cm*Um* 2′-OMe, and the firstGm*Cm*Gm (SEQ ID NO: 361) and last 5 internucleotide linkages werephosphorothioated HIV2-71-CM1 55nt-c- CM1:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 15ntModifications in CM0Um-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G- and all U: are withA-C-A-C-Um-Um-C-G-G-C-C-C-A-G-A-G-C-Um-G-C-Um-C-C-U 2′-OMem*Cm*Am*Um (SEQ ID NO: 350) HIV2-71-CM2 55nt-c- CM2:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 15ntModifications in CM1Um-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G- and the targetingA-C-A-C-Um-Um-C-G-G-C-C-C-Am-G-A-G-C-Um-G-C-Um-C-C- triplet is CCAmUm*Cm*Am*Um (SEQ ID NO: 350) HIV2-71-CM3 55nt-c- CM3:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 15nt Modifications inUm-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G-CM1 and the targetingA-C-A-C-Um-Um-C-G-G-C-Cm-C-A-G-A-G-C-Um-G-C-Um-C-C- triplet is CmCAUm*Cm*Am*Um (SEQ ID NO: 350) HIV2-71-CM4 55nt-c- CM4:Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um-Um-G-C- 15ntModifications in CM1Um-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G- and the targetingA-C-A-C-Um-Um-C-G-G-C-C*C*A*G-A-G-C-Um-G-C-Um-C-C- triplet is C*C*A*Um*Cm*Am*Um (SEQ ID NO: 350) HIV2-71-CM5 55nt-c- CM5:Gm*Am*Cm*Gm-Cm-Cm-Cm-Am-Cm-Cm-Gm-Um-Gm-Um-Gm- 15nt Modifications in CM1Gm-Um-Um-Gm-Cm-Um-Gm-Um-Cm-Cm-Am-Gm-Gm-Am-Cm- and all nucleotides areGm-Gm-Um-Cm-Cm-Cm-Gm-Gm-Cm-Cm-Um-Gm-Cm-Gm-Am- with 2′-OMe, exceptCm-Am-Cm-Um-Um-C-G-G-C-C-C-A-G-A-G-C-Um-Gm-Cm-Um- for the targetingCm-Cm-Um*Cm*Am*Um (SEQ ID NO: 350) nucleotide and 5nt on each side of itHIV2-71-CM6 55nt-c- CM6:Gm*Am*Cm*Gm*Cm*C-C-A-C-C-G-U-G-U-G-G-U-U-G-C-U-G- 15nt5 terminal bases atU-C-C-A-G-G-A-C-G-G-U-C-C-C-G-G-C-C-U-G-C-G-A-C-A-C-U- each terminus areU-C-G-G-C-C-C-A-G-A-G-C-U-G-C-U-C*Cm*Um*Cm*Am*Um with 2′-OMe and the(SEQ ID NO: 350) first and last 5 internucleotide linkages werephosphorothioated Note: “m” refers to 2′-O-Me on the ribose of thenucleotide “*” refers to phosphorothioate linkage

Example 29. Further Verification of the Modification Pattern

The modification pattern of CM1 was tested on another sequence. Apreferable modification pattern in a prior art was used as a control. Asshown in table 14, 55 nt-c-15 nt-CM1 was the test sequence, and 36nt-c-13 nt-CM11 was a positive control, in which, all the nucleotides,except for the editing triplet “CCA”, are modified with 2′-O-Me, and thefirst and last 4 internucleotide linkages were phosphorothioated. Inaddition, 36 nt-c-13 nt-CM11 was only 51 nt, which is not a preferablelength in this invention but a preferable length in the prior art. 48hours after the transfection of the dRNAs into GM06214 cells usingLipofectamine RNAiMAX, IDUA enzyme activity was detected using themethod shown in Example 23. As shown in FIG. 44, 55 nt-c-15 nt-CM1 had asignificantly higher editing efficiency than that of 36 nt-c-13 nt-CM11.

TABLE 14 Name Modification pattern Sequence 55nt-c-15nt- CM1Gm*Am*Cm*G-C-C-C-A-C-C-G-Um-G-Um-G-G-Um- CM1Um-G-C-Um-G-Um-C-C-A-G-G-A-C-G-G-Um-C-C-C-G-G-C-C-Um-G-C-G-A-C-A-C-Um-Um-C-G-G-C-C-C-A-G-A-G-C-Um-G-C-Um-C-C-Um*Cm*Am*Um (SEQ ID NO: 366) 36nt-c-13nt-CM11: Cm*Um*Gm*Um*Cm-Cm-Am-Gm-Gm-Am-Cm-Gm- CM11All nucleotides, except for the targetingGm-Um-Cm-Cm-Cm-Gm-Gm-Cm-Cm-Um-Gm-Cm-triplet CroCroAro, are modified withGm-Am-Cm-Am-Cm-Um-Um-Cm-Gm-Gm-Cm-C-C-A- 2′-O-Me, the first and last 4Gm-Am-Gm-Cm-Um-Gm-Cm-Um*Cm*Cm*Um*Cm internucleotide linkages were(SEQ ID NO: 414) phosphorothioated Note: “m” refers to 2′-O-Me on theribose of the nucleotide. ”*” refers to phosphorothioate linkage

Example 30. Further Test of the dRNAs in Other Cells

This example focused on the repair of USH2A c.11864 G>A (p.Trp3955*)mutation using LEAPER technology. The reporter system designed in thisexample is shown in FIG. 45A. In the case of USH2A c.11864 G>A(p.Trp3955*, the normal TGG sequence was mutated to TAG which is a stopcodon. Thus, translation of the mutated mRNA will be terminated early atthis TAG. The 293T (293T cells from C. Zhang's laboratory, PekingUniversity) reporter system is a lentiviral vector, and the mRNA shownin FIG. 45A above is driven by a CMV promoter. The system comprises thefollowing parts: 1) mCherry red fluorescent protein, which can be stablyexpressed, 2) the mutation site of USH2A gene and the adjacent 100 basepairs on both sides. 3) GFP green fluorescent protein. When the mutationsite is successfully edited, the TAG codon is converted TIG, whichallows translation to continue, and the GFP after the USH2A sequence canbe translated normally. Thus, the expression of GFP represents theediting efficiency.

The dRNA were synthesized in vitro, and all the dRNA sequences used inthis example were shown in Table 15. All the dRNA sequences weremodified in CM0 pattern. The specific steps of the test were as follows:

293T reporter cells were cultured in DMEM (Hyclone SH30243.01) with 10%FBS (Vistech, SE100-011). When confluent, cells were transferred into 12well plates at 15,000 cells/well. The time is recorded as 0 hr.

At 24 hr, 293T cells in each well were transfected with 12.5 pmol ofdRNA using Lipofectamine RNAiMAX reagent (Invitrogen 13778150).Transfection protocol was provided in the product manual.

At 72 hr, cells in each well were digested with trypsin (Invitrogen,13778-150), and the intensity of FITC (Fluorescein isothiocyanate) wasdetected using a flow cytometer.

As shown in FIG. 45B, cells were the editing efficiency of dRNAs with 3′and 5′ termini of equal length. NC represents the control cells withoutdRNA transfection. In accordance with the above examples, the GFPpositive ratio of the cells transfected with dRNAs of 111 nt, 91 nt and71 nt exceed 90%, while cells transfected with 51 nt dRNA resulted in avery low GFP positive ratio. From the data of MFI (mean fluorescenceintensity) on the left, the 111 nt dRNA led to the highest fluorescenceintensity.

As shown in FIG. 45C, dRNAs with 3′ and 5′ termini of different lengthsand a 111 nt dRNA with equal 3′ and 5′ termini were transfected intocells, separately. As used in this example, the dRNA with a 55 nt 5′terminus has a 3′ terminus of 55 nt, 45 nt, 35 nt, 25 nt, or 5 nt.Similarly, the dRNA with a 55 nt 3′ terminus has a 5′ terminus of 55 nt,45 nt, 35 nt, 25 nt, or 5 nt. According to the result in FIG. 45C, theediting efficiency decreased dramatically when the length of dRNA wasreduced to 61 nt, while the longer dRNAs had obviously higher editingefficiency. Among them, dRNA 55 nt-c-25 nt had the highest editingefficiency. Thus, the 3′ terminus was fixed to 25 nt, and dRNAs with 5′termini of different lengths from 55 nt to 25 nt. The result of cellstransfected with these dRNAs was shown in FIG. 45D. Two 55 nt-c-25 ntdRNAs were from 2 different batches. It was obvious that the shorter the5′ terminus, the lower the editing efficiency. In addition, result inFIG. 45 Donce again indicated that, to ensure the editing efficiency,the length of dRNA is preferably not to be less than 61 nt.

TABLE 15 Sequence Length No. Sequence 55nt-C- SEQ IDagcccaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguua55nt NO: 414 caggcucugacccgauauucguagag 45nt-C- SEQ IDgcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcucuga45nt NO: 415 cccgau 35nt-C- SEQ IDcuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcu35nt NO: 416 25nt-C- SEQ IDagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga 25nt NO: 417 55nt-C-SEQ IDagcccaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguua45nt NO: 418 caggcucugacccgau 55nt-C- SEQ IDagcccaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguua35nt NO: 419 caggcu 55nt-C- SEQ IDagcccaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga25nt NO: 420 55nt-C- SEQ IDagcccaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacug15nt NO: 421 55nt-C- SEQ IDagcccaaggagcuggaaaaucuugaggaggagcuuccagaguuuguguuaaugaccacaga 5ntNO: 422 45nt-C- SEQ IDgcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcucuga55nt NO: 423 cccgauauucguagag 35nt-C- SEQ IDcuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcucugacccgauauuc55nt NO: 424 guagag 25nt-C- SEQ IDagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcucugacccgauauucguagag55nt NO: 425 15nt-C- SEQ IDguuuguguuaaugaccacagacucuccacugaacccuuggaguuacaggcucugacccgauauucguagag55nt NO: 426 5nt-C- SEQ IDaugaccacagacucuccacugaacccuuggaguuacaggcucugacccgauauucguagag 55ntNO: 427 50nt-C- SEQ IDaaggagcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga25nt NO: 428 45nt-C- SEQ IDgcuggaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga25nt NO: 429 40-C- SEQ IDaaaaucuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga 25ntNO: 430 35nt-C- SEQ IDcuugagguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga 25ntNO: 431 30-C- SEQ IDgguggagcuuccagaguuuguguuaaugaccacagacucuccacugaacccuugga 25nt NO: 432

ADAR1(p110) cDNA (SEQ ID NO: 332)5′-atggccgagatcaaggagaaaatctgcgactatctcttcaatgtgtctgactcctctgccctgaatttggctaaaaatattggccttaccaaggcccgagatataaatgctgtgctaattgacatggaaaggcagggggatgtctatagacaagggacaacccctcccatatggcatttgacagacaagaagcgagagaggatgcaaatcaagagaaatacgaacagtgttcctgaaaccgctccagctgcaatccctgagaccaaaagaaacgcagagttcctcacctgtaatatacccacatcaaatgcctcaaataacatggtaaccacagaaaaagtggagaatgggcaggaacctgtcataaagttagaaaacaggcaagaggccagaccagaaccagcaagactgaaaccacctgttcattacaatggcccctcaaaagcagggtatgttgactttgaaaatggccagtgggccacagatgacatcccagatgacttgaatagtatccgcgcagcaccaggtgagtttcgagccatcatggagatgccctccttctacagtcatggcttgccacggtgttcaccctacaagaaactgacagagtgccagctgaagaaccccatcagcgggctgttagaatatgcccagttcgctagtcaaacctgtgagttcaacatgatagagcagagtggaccaccccatgaacctcgatttaaattccaggttgtcatcaatggccgagagtttcccccagctgaagctggaagcaagaaagtggccaagcaggatgcagctatgaaagccatgacaattctgctagaggaagccaaagccaaggacagtggaaaatcagaagaatcatcccactattccacagagaaagaatcagagaagactgcagagtcccagacccccaccccttcagccacatccttcttttctgggaagagccccgtcaccacactgcttgagtgtatgcacaaattggggaactcctgcgaattccgtctcctgtccaaagaaggccctgcccatgaacccaagttccaatactgtgttgcagtgggagcccaaactttccccagtgtgagtgctcccagcaagaaagtggcaaagcagatggccgcagaggaagccatgaaggccctgcatggggaggcgaccaactccatggcttctgataaccagcctgaaggtatgatctcagagtcacttgataacttggaatccatgatgcccaacaaggtcaggaagattggcgagctcgtgagatacctgaacaccaaccctgtgggtggccttttggagtacgcccgctcccatggctttgctgctgaattcaagttggtcgaccagtccggacctcctcacgagcccaagttcgtttaccaagcaaaagttgggggtcgctggttcccagccgtctgcgcacacagcaagaagcaaggcaagcaggaagcagcagatgcggctctccgtgtcttgattggggagaacgagaaggcagaacgcatgggtttcacagaggtaaccccagtgacaggggccagtctcagaagaactatgctcctcctctcaaggtccccagaagcacagccaaagacactccctctcactggcagcaccttccatgaccagatagccatgctgagccaccggtgcttcaacactctgactaacagcttccagccctccttgctcggccgcaagattctggccgccatcattatgaaaaaagactctgaggacatgggtgtcgtcgtcagcttgggaacagggaatcgctgtgtaaaaggagattctctcagcctaaaaggagaaactgtcaatgactgccatgcagaaataatctcccggagaggcttcatcaggtttctctacagtgagttaatgaaatacaactcccagactgcgaaggatagtatatttgaacctgctaagggaggagaaaagctccaaataaaaaagactgtgtcattccatctgtatatcagcactgctccgtgtggagatggcgccctctttgacaagtcctgcagcgaccgtgctatggaaagcacagaatcccgccactaccctgtcttcgagaatcccaaacaaggaaagctccgcaccaaggtggagaacggagaaggcacaatccctgtggaatccagtgacattgtgcctacgtgggatggcattcggctcggggagagactccgtaccatgtcctgtagtgacaaaatcctacgctggaacgtgctgggcctgcaaggggcactgttgacccacttcctgcagcccatttatctcaaatctgtcacattgggttaccttttcagccaagggcatctgacccgtgctatttgctgtcgtgtgacaagagatgggagtgcatttgaggatggactacgacatccctttattgtcaaccaccccaaggttggcagagtcagcatatatgattccaaaaggcaatccgggaagactaaggagacaagcgtcaactggtgtctggctgatggctatgacctggagatcctggacggtaccagaggcactgtggatgggccacggaatgaattgtcccgggtctccaaaaagaacatttttcttctatttaagaagctctgctccttccgttaccgcagggatctactgagactctcctatggtgaggccaagaaagctgcccgtgactacgagacggccaagaactacttcaaaaaaggcctgaaggatatgggctatgggaactggattagcaaaccccaggaggaaaagaacttttatctctgcccagta gattacaaggatgacgacgataag(Flag tag) TAG-3′ ADAR1(p150) cDNA (SEQ ID NO: 333)5′atgaatccgcggcaggggtattccctcagcggatactacacccatccatttcaaggctatgagcacagacagctcagataccagcagcctgggccaggatcttcccccagtagtttcctgcttaagcaaatagaatttctcaaggggcagctcccagaagcaccggtgattggaaagcagacaccgtcactgccaccttccctcccaggactccggccaaggtttccagtactacttgcctccagtaccagaggcaggcaagtggacatcaggggtgtccccaggggcgtgcatctcggaagtcaggggctccagagagggttccagcatccttcaccacgtggcaggagtctgccacagagaggtgttgattgcctttcctcacatttccaggaactgagtatctaccaagatcaggaacaaaggatcttaaagttcctggaagagcttggggaagggaaggccaccacagcacatgatctgtctgggaaacttgggactccgaagaaagaaatcaatcgagttttatactccctggcaaagaagggcaagctacagaaagaggcaggaacaccccctttgtggaaaatcgcggtctccactcaggcttggaaccagcacagcggagtggtaagaccagacggtcatagccaaggagccccaaactcagacccgagtttggaaccggaagacagaaactccacatctgtctcagaagatcttcttgagccttttattgcagtctcagctcaggcttggaaccagcacagcggagtggtaagaccagacagtcatagccaaggatccccaaactcagacccaggtttggaacctgaagacagcaactccacatctgccttggaagatcctcttgagtttttagacatggccgagatcaaggagaaaatctgcgactatctcttcaatgtgtctgactcactgccctgaatttggctaaaaatattggccttaccaaggcccgagatataaatgctgtgctaattgacatggaaaggcagggggatgtctatagacaagggacaacccctcccatatggcatttgacagacaagaagcgagagaggatgcaaatcaagagaaatacgaacagtgttcctgaaaccgctccagctgcaatccctgagaccaaaagaaacgcagagttcctcacctgtaatatacccacatcaaatgcctcaaataacatggtaaccacagaaaaagtggagaatgggcaggaacctgtcataaagttagaaaacaggcaagaggccagaccagaaccagcaagactgaaaccacctgttcattacaatggcccctcaaaagcagggtatgttgactttgaaaatggccagtgggccacagatgacatcccagatgacttgaatagtatccgcgcagcaccaggtgagtttcgagccatcatggagatgccctccttctacagtcatggcttgccacggtgttcaccctacaagaaactgacagagtgccagctgaagaaccccatcagcgggctgttagaatatgcccagttcgctagtcaaacctgtgagttcaacatgatagagcagagtggaccaccccatgaacctcgatttaaattccaggttgtcatcaatggccgagagtttcccccagctgaagctggaagcaagaaagtggccaagcaggatgcagctatgaaagccatgacaattctgctagaggaagccaaagccaaggacagtggaaaatcagaagaatcatcccactattccacagagaaagaatcagagaagactgcagagtcccagacccccaccccttcagccacatccttcttttctgggaagagccccgtcaccacactgcttgagtgtatgcacaaattggggaactcctgcgaattccgtctcctgtccaaagaaggccctgcccatgaacccaagttccaatactgtgttgcagtgggagcccaaactttccccagtgtgagtgctcccagcaagaaagtggcaaagcagatggccgcagaggaagccatgaaggccctgcatggggaggcgaccaactccatggcttctgataaccagcctgaaggtatgatctcagagtcacttgataacttggaatccatgatgcccaacaaggtcaggaagattggcgagctcgtgagatacctgaacaccaaccctgtgggtggccttttggagtacgcccgctcccatggctttgctgctgaattcaagttggtcgaccagtccggacctcctcacgagcccaagttcgtttaccaagcaaaagttgggggtcgctggttcccagccgtctgcgcacacagcaagaagcaaggcaagcaggaagcagcagatgcggctctccgtgtcttgattggggagaacgagaaggcagaacgcatgggtttcacagaggtaaccccagtgacaggggccagtctcagaagaactatgctcctcctctcaaggtccccagaagcacagccaaagacactccctctcactggcagcaccttccatgaccagatagccatgctgagccaccggtgcttcaacactctgactaacagcttccagccctccttgctcggccgcaagattctggccgccatcattatgaaaaaagactctgaggacatgggtgtcgtcgtcagcttgggaacagggaatcgctgtgtaaaaggagattctctcagcctaaaaggagaaactgtcaatgactgccatgcagaaataatctcccggagaggcttcatcaggtttctctacagtgagttaatgaaatacaactcccagactgcgaaggatagtatatttgaacctgctaagggaggagaaaagctccaaataaaaaagactgtgtcattccatctgtatatcagcactgctccgtgtggagatggcgccctctttgacaagtcctgcagcgaccgtgctatggaaagcacagaatcccgccactaccctgtcttcgagaatcccaaacaaggaaagctccgcaccaaggtggagaacggagaaggcacaatccctgtggaatccagtgacattgtgcctacgtgggatggcattcggctcggggagagactccgtaccatgtcctgtagtgacaaaatcctacgctggaacgtgctgggcctgcaaggggcactgttgacccacttcctgcagcccatttatctcaaatctgtcacattgggttaccttttcagccaagggcatctgacccgtgctatttgctgtcgtgtgacaagagatgggagtgcatttgaggatggactacgacatccctttattgtcaaccaccccaaggttggcagagtcagcatatatgattccaaaaggcaatccgggaagactaaggagacaagcgtcaactggtgtctggctgatggctatgacctggagatcctggacggtaccagaggcactgtggatgggccacggaatgaattgtcccgggtctccaaaaagaacatttttcttctatttaagaagctctgctccttccgttaccgcagggatctactgagactctcctatggtgaggccaagaaagctgcccgtgactacgagacggccaagaactacttcaaaaaaggcctgaaggatatgggctatgggaactggattagcaaaccccaggaggaaaagaacttttatctctgcccagta gattacaaggatgacgacgataag(Flag tag) TAG-3′ADAR2 cDNA (seq id no: 334)5′-atggatatagaagatgaagaaaacatgagttccagcagcactgatgtgaaggaaaaccgcaatctggacaacgtgtcccccaaggatggcagcacacctgggcctggcgagggctctcagctctccaatgggggtggtggtggccccggcagaaagcggcccctggaggagggcagcaatggccactccaagtaccgcctgaagaaaaggaggaaaacaccagggcccgtcctccccaagaacgccctgatgcagctgaatgagatcaagcctggtttgcagtacacactcctgtcccagactgggcccgtgcacgcgcctttgtttgtcatgtctgtggaggtgaatggccaggtttttgagggctctggtcccacaaagaaaaaggcaaaactccatgctgctgagaaggccttgaggtattcgttcagtttcctaatgcctctgaggcccacctggccatggggaggaccctgtctgtcaacacggacttcacatctgaccaggccgacttccctgacacgctcttcaatggttttgaaactcctgacaaggcggagcctcccttttacgtgggctccaatggggatgactccttcagttccagcggggacctcagcttgtctgcttccccggtgcctgccagcctagcccagcctcctctccctgccttaccaccattcccacccccgagtgggaagaatcccgtgatgatcttgaacgaactgcgcccaggactcaagtatgacttcctctccgagagcggggagagccatgccaagagcttcgtcatgtctgtggtcgtggatggtcagttctttgaaggctcggggagaaacaagaagcttgccaaggcccgggctgcgcagtctgccctggccgccatttttaacttgcacttggatcagacgccatctcgccagcctattcccagtgagggtcttcagctgcatttaccgcaggttttagctgacgctgtctcacgcctggtcctgggtaagtttggtgacctgaccgacaacttctcctcccctcacgctcgcagaaaagtgctggctggagtcgtcatgacaacaggcacagatgttaaagatgccaaggtgataagtgtttctacaggaacaaaatgtattaatggtgaatacatgagtgatcgtggccttgcattaaatgactgccatgcagaaataatatctcggagatccttgctcagatttctttatacacaacttgagctttacttaaataacaaagatgatcaaaaaagatccatctttcagaaatcagagcgaggggggtttaggctgaaggagaatgtccagtttcatctgtacatcagcacctctccctgtggagatgccagaatcttctcaccacatgagccaatcctggaagaaccagcagatagacacccaaatcgtaaagcaagaggacagctacggaccaaaatagagtctggtgaggggacgattccagtgcgctccaatgcgagcatccaaacgtgggacggggtgctgcaaggggagcggctgctcaccatgtcctgcagtgacaagattgcacgctggaacgtggtgggcatccagggatccctgctcagcattttcgtggagcccatttacttctcgagcatcatcctgggcagcctttaccacggggaccacctttccagggccatgtaccagcggatctccaacatagaggacctgccacctctctacaccctcaacaagcattgctcagtggcatcagcaatgcagaagcacggcagccagggaaggcccccaacttcagtgtcaactggacggtaggcgactccgctattgaggtcatcaacgccacgactgggaaggatgagctgggccgcgcgtcccgcctgtgtaagcacgcgttgtactgtcgctggatgcgtgtgcacggcaaggttccctcccacttactacgctccaagattaccaaacccaacgtgtaccatgagtccaagctggcggcaaaggagtaccaggccgccaaggcgcgtctgttcacagccttcatcaaggcggggctgggggcctgggtggagaagcccaccgagcaggaccagttctcactcacgccc gattacaaggatgacgacgataag(flag tag) tag-3′Coding sequence (CDS) of the disease-relevant genes COL3A1(SEQ ID NO: 335)5′-atgatgagctttgtgcaaaaggggagctggctacttctcgctctgcttcatcccactattattttggcacaacaggaagctgttgaaggaggatgttcccatcttggtcagtcctatgcggatagagatgtctggaagccagaaccatgccaaatatgtgtctgtgactcaggatccgttctctgcgatgacataatatgtgacgatcaagaattagactgccccaacccagaaattccatttggagaatgttgtgcagtttgcccacagcctccaactgctcctactcgccctcctaatggtcaaggacctcaaggccccaagggagatccaggccctcctggtattcctgggagaaatggtgaccctggtattccaggacaaccagggtcccctggttctcctggcccccctggaatctgtgaatcatgccctactggtcctcagaactattctccccagtatgattcatatgatgtcaagtctggagtagcagtaggaggactcgcaggctatcctggaccagctggccccccaggccctcccggtccccctggtacatctggtcatcctggttcccctggatctccaggataccaaggaccccctggtgaacctgggcaagctggtccttcaggccctccaggacctcctggtgctataggtccatctggtcctgctggaaaagatggagaatcaggtagacccggacgacctggagagcgaggattgcctggacctccaggtatcaaaggtccagctgggatacctggattccctggtatgaaaggacacagaggcttcgatggacgaaatggagaaaagggtgaaacaggtgctcctggattaaagggtgaaaatggtcttccaggcgaaaatggagctcctggacccatgggtccaagaggggctcctggtgagcgaggacggccaggacttcctggggctgcaggtgctcggggtaatgacggtgctcgaggcagtgatggtcaaccaggccctcctggtcctcctggaactgccggattccctggatcccctggtgctaagggtgaagttggacctgcagggtctcctggttcaaatggtgcccctggacaaagaggagaacctggacctcagggacacgctggtgctcaaggtcctcctggccctcctgggattaatggtagtcctggtggtaaaggcgaaatgggtcccgctggcattcctggagctcctggactgatgggagcccggggtcctccaggaccagccggtgctaatggtgctcctggactgcgaggtggtgcaggtgagcctggtaagaatggtgccaaaggagagcccggaccacgtggtgaacgcggtgaggctggtattccaggtgttccaggagctaaaggcgaagatggcaaggatggatcacctggagaacctggtgcaaatgggcttccaggagctgcaggagaaaggggtgcccctgggttccgaggacctgctggaccaaatggcatcccaggagaaaagggtcctgaggagagcgtggtgctccaggccctgcagggcccagaggagctgctggagaacctggcagagatggcgtccctggaggtccaggaatgaggggcatgcccggaagtccaggaggaccaggaagtgatgggaaaccagggcctcccggaagtcaaggagaaagtggtcgaccaggtcctcctgggccatctggtccccgaggtcagcctggtgtcatgggcttccccggtcctaaaggaaatgatggtgctcctggtaagaatggagaacgaggtggccctggaggacctggccctcagggtcctcctggaaagaatggtgaaactggacctcagggacccccagggcctactgggcctggtggtgacaaaggagacacaggaccccctggtccacaaggattacaaggcttgcctggtacaggtggtcctccaggagaaaatggaaaacctggggaaccaggtccaaagggtgatgccggtgcacctggagctccaggaggcaagggtgatgctggtgcccctggtgaacgtggacctcctggattggcaggggccccaggacttagaggtggagctggtccccctggtcccgaaggaggaaagggtgctgctggtcctcctgggccacctggtgctgctggtactcctggtctgcaaggaatgcctggagaaagaggaggtcttggaagtcctggtccaaagggtgacaagggtgaaccaggcggtccaggtgctgatggtgtcccagggaaagatggcccaaggggtcctactggtcctattggtcctcctggcccagctggccagcctggagataagggtgaaggtggtgcccccggacttccaggtatagctggacctcgtggtagccctggtgagagaggtgaaactggccctccaggacctgctggtttccctggtgctcctggacagaatggtgaacctggtggtaaaggagaaagaggggctccgggtgagaaaggtgaaggaggccctcctggagttgcaggaccccctggaggttctggacctgctggtcctcctggtccccaaggtgtcaaaggtgaacgtggcagtcctggtggacctggtgctgctggcttccctggtgctcgtggtcttcctggtcctcctggtagtaatggtaacccaggacccccaggtcccacgggttctccaggcaaggatgggcccccaggtcctgcgggtaacactggtgctcctggcagccctggagtgtctggaccaaaaggtgatgctggccaaccaggagagaagggatcgcctggtgcccagggcccaccaggagctccaggcccacttgggattgctgggatcactggagcacggggtcttgcaggaccaccaggcatgccaggtcctaggggaagccctggccctcagggtgtcaagggtgaaagtgggaaaccaggagctaacggtctcagtggagaacgtggtccccctggaccccagggtcttcctggtctggctggtacagctggtgaacctggaagagatggaaaccctggatcagatggtcttccaggccgagatggatctcctggtggcaagggtgatcgtggtgaaaatggctctcctggtgcccctggcgctcctggtcatccaggcccacctggtcctgtcggtccagctggaaagagtggtgacagaggagaaagtggccctgctggccctgctggtgctcccggtcctgctggttcccgaggtgctcctggtcctcaaggcccacgtggtgacaaaggtgaaacaggtgaacgtggagctgctggcatcaaaggacatcgaggattccctggtaatccaggtgccccaggttctccaggccctgctggtcagcagggtgcaatcggcagtccaggacctgcaggccccagaggacctgttggacccagtggacctcctggcaaagatggaaccagtggacatccaggtcccattggaccaccagggcctcgaggtaacagaggtgaaagaggatctgagggctccccaggccacccagggcaaccaggccctcctggacctcctggtgcccctggtccttgctgtggtggtgttggagccgctgccattgctgggattggaggtgaaaaagctggcggttttgccccgtattatggagatgaaccaatggatttcaaaatcaacaccgatgagattatgacttcactcaagtctgttaatggacaaatagaaagcctcattagtcctgatggttctcgtaaaaaccccgctagaaactgcagagacctgaaattctgccatcctgaactcaagagtggagaatactgggttgaccctaaccaaggatgcaaattggatgctatcaaggtattctgtaatatggaaactggggaaacatgcataagtgccaatcctttgaatgttccacggaaacactggtggacagattctagtgctgagaagaaacacgtttggtttggagagtccatggatggtggttttcagtttagctacggcaatcctgaacttcctgaagatgtccttgatgtgcagctggcattccttcgacttctctccagccgagcttcccagaacatcacatatcactgcaaaaatagcattgcatacatggatcaggccagtggaaatgtaaagaaggccctgaagctgatggggtcaaatgaaggtgaattcaaggctgaaggaaatagcaaattcacctacacagttctggaggatggttgcacgaaacacactggggaatggagcaaaacagtctttgaatatcgaacacgcaaggctgtgagactacctattgtagatattgcaccctatgacattggtggtcctgatcaagaatttggtgtggacgttggccctgtttgctttttataa-3′ BMPR2(SEQ ID NO: 336)5′-atgacttcctcgagcagcggccaggcgggtgccaggctaccatggaccatcctgctggtcagcgagcggctgcttcgcagaatcaagaacggctatgtgcgtttaaagatccgtatcagcaagaccttgggataggtgagagtagaatctctcatgaaaatgggacaatattatgctcgaaaggtagcacctgctatggcctttgggagaaatcaaaaggggacataaatcttgtaaaacaaggatgttggtctcacattggagatccccaagagtgtcactatgaagaatgtgtagtaactaccactcctccctcaattcagaatggaacataccgtttctgctgttgtagcacagatttatgtaatgtcaactttactgagaattttccacctcctgacacaacaccactcagtccacctcattcatttaaccgagatgagacaataatcattgattggcatcagtctctgtattagctgttttgatagttgccttatgctttggatacagaatgttgacaggagaccgtaaacaaggtcttcacagtatgaacatgatggaggcagcagcatccgaaccctctcttgatctagataatctgaaactgttggagctgattggccgaggtcgatatggagcagtatataaaggctccttggatgagcgtccagttgctgtaaaagtgttttcctttgcaaaccgtcagaattttatcaacgaaaagaacatttacagagtgcctttgatggaacatgacaacattgcccgctttatagttggagatgagagagtcactgcagatggacgcatggaatatttgcttgtgatggagtactatcccaatggatctttatgcaagtatttaagtctccacacaagtgactgggtaagctcttgccgtcttgctcattctgttactagaggactggcttatcttcacacagaattaccacgaggagatcattataaacctgcaatttcccatcgagatttaaacagcagaaatgtcctagtgaaaaatgatggaacctgtgttattagtgactttggactgtccatgaggctgactggaaatagactggtgcgcccaggggaggaagataatgcagccataagcgaggttggcactatcagatatatggcaccagaagtgctagaaggagctgtgaacttgagggactgtgaatcagctttgaaacaagtagacatgtatgctcttggactaatctattgggagatatttatgagatgtacagacctcttcccaggggaatccgtaccagagtaccagatggcttttcagacagaggttggaaaccatcccacttttgaggatatgcaggttctcgtgtctagggaaaaacagagacccaagttcccagaagcctggaaagaaaatagcctggcagtgaggtcactcaaggagacaatcgaagactgttgggaccaggatgcagaggctcggcttactgcacagtgtgctgaggaaaggatggctgaacttatgatgatttgggaaagaaacaaatctgtgagcccaacagtcaatccaatgtctactgctatgcagaatgaacgcaacctgtcacataataggcgtgtgccaaaaattggtccttatccagattattcttcctcctcatacattgaagactctatccatcatactgacagcatcgtgaagaatatttcctctgagcattctatgtccagcacacctttgactataggggaaaaaaaccgaaattcaattaactatgaacgacagcaagcacaagctcgaatccccagccctgaaacaagtgtcaccagcctctccaccaacacaacaaccacaaacaccacaggactcacgccaagtactggcatgactactatatctgagatgccatacccagatgaaacaaatctgcataccacaaatgttgcacagtcaattgggccaacccctgtctgcttacagctgacagaagaagacttggaaaccaacaagctagacccaaaagaagttgataagaacctcaaggaaagctctgatgagaatctcatggagcactctcttaaacagttcagtggcccagacccactgagcagtactagttctagcttgctttacccactcataaaacttgcagtagaagcaactggacagcaggacttcacacagactgcaaatggccaagcatgtttgattcctgatgttctgcctactcagatctatcctctccccaagcagcagaaccttcccaagagacctactagtttgcctttgaacaccaaaaattcaacaaaagagccccggctaaaatttggcagcaagcacaaatcaaacttgaaacaagtcgaaactggagttgccaagatgaatacaatcaatgcagcagaacctcatgtggtgacagtcaccatgaatggtgtggcaggtagaaaccacagtgttaactcccatgctgccacaacccaatatgccaatgggacagtactatctggccaaacaaccaacatagtgacacatagggcccaagaaatgttgcagaatcagtttattggtgaggacacccggctgaatattaattccagtcctgatgagcatgagcctttactgagacgagagcaacaagctggccatgatgaaggtgttctggatcgtcttgtggacaggagggaacggccactagaaggtggccgaactaattccaataacaacaacagcaatccatgttcagaacaagatgttcttgcacagggtgttccaagcacagcagcagatcctgggccatcaaagcccagaagagcacagaggcctaattctctggatctttcagccacaaatgtcctggatggcagcagtatacagataggtgagtcaacacaagatggcaaatcaggatcaggtgaaaagatcaagaaacgtgtgaaaactccctattctcttaagcggtggcgcccctccacctgggtcatctccactgaatcgctggactgtgaagtcaacaataatggcagtaacagggcagttcattccaaatccagcactgctgtttaccttgcagaaggaggcactgctacaaccatggtgtctaaagatataggaatgaactgtctgtga-3′AHI1 (SEQ ID NO: 337)5′-atgcctaagctgagagtgaagcaaaagtaaaaaccaaagttcgctttgaagaattgcttaagacccacagtgatctaatgcgtgaaaagaaaaaactgaagaaaaaacttgtcaggtctgaagaaaacatctcacctgacactattagaagcaatcttcactatatgaaagaaactacaagtgatgatcccgacactattagaagcaatcttccccatattaaagaaactacaagtgatgatgtaagtgctgctaacactaacaacctgaagaagagcacgagagtcactaaaaacaaattgaggaacacacagttagcaactgaaaatcctaatggtgatgctagtgtagaggaagacaaacaaggaaagccaaataaaaaggtgataaagacggtgccccagttgactacacaagacctgaaaccggaaactcctgagaataaggttgattctacacaccagaaaacacatacaaagccacagccaggcgttgatcatcagaaaagtgagaaggcaaatgagggaagagaagagactgatttagaagaggatgaagaattgatgcaagcatatcagtgccatgtaactgaagaaatggcaaaggagattaagaggaaaataagaaagaaactgaaagaacagttgacttactttccctcagatactttattccatgatgacaaactaagcagtgaaaaaaggaaaaagaaaaaggaagttccagtcttctctaaagctgaaacaagtacattgaccatctctggtgacacagttgaaggtgaacaaaagaaagaatcttcagttagatcagtttcttcagattctcatcaagatgatgaaataagctcaatggaacaaagcacagaagacagcatgcaagatgatacaaaacctaaaccaaaaaaaacaaaaaagaagactaaagcagttgcagataataatgaagatgttgatggtgatggtgttcatgaaataacaagccgagatagcccggtttatcccaaatgtttgtcttgatgatgaccttgtcttgggagtttacattcaccgaactgatagacttaagtcagattttatgatttctcacccaatggtaaaaattcatgtggttgatgagcatactggtcaatatgtcaagaaagatgatagtggacggcctgtttcatcttactatgaaaaagagaatgtggattatattcttcctattatgacccagccatatgattttaaacagttaaaatcaagacttccagagtgggaagaacaaattgtatttaatgaaaattttccctatttgcttcgaggctctgatgagagtcctaaagtcatcctgttctttgagattcttgatttcttaagcgtggatgaaattaagaataattctgaggttcaaaaccaagaatgtggctttcggaaaattgcctgggcatttataagatctgggagccaatggaaatgcaaacatcaactcaaaacttcgcttgcagctatattacccacctactaagcctcgatccccattaagtgttgttgaggcatttgaatggtggtcaaaatgtccaagaaatcattacccatcaacactgtacgtaactgtaagaggactgaaagttccagactgtataaagccatcttaccgctctatgatggctcttcaggaggaaaaaggtaaaccagtgcattgtgaacgtcaccatgagtcaagacagtagacacagaacctggattagaagagtcaaaggaagtaataaagtggaaacgactccctgggcaggcttgccgtatcccaaacaaacacctcttctcactaaatgcaggagaacgaggatgtttttgtcttgatttctcccacaatggaagaatattagcagcagcttgtgccagccgggatggatatccaattattttatatgaaattccttctggacgtttcatgagagaattgtgtggccacctcaatatcatttatgatctttcctggtcaaaagatgatcactacatccttacttcatcatctgatggcactgccaggatatggaaaaatgaaataaaraatacaaatactttcagagttttacctcatccttcttttgtttacacggctaaattccatccagctgtaagagagctagtagttacaggatgctatgattccatgatacggatatggaaagttgagatgagagaagattctgccatattggtccgacagtttgacgttcacaaaagttttatcaactcactttgttttgatactgaaggtcatcatatgtattcaggagattgtacaggggtgattgttgtttggaatacctatgtcaagattaatgatttggaacattcagtgcaccactggactataaataaggaaattaaagaaactgagtttaagggaattccaataagttatttggagattcatcccaatggaaaacgtttgttaatccataccaaagacagtactttgagaattatggatctccggatattagtagcaaggaagtttgtaggagcagcaaattatcgggagaagattcatagtactttgactccatgtgggacttttctgtttgctggaagtgaggatggtatagtgtatgtttggaacccagaaacaggagaacaagtagccatgtattctgacttgccattcaagtcacccattcgagacatttcttatcatccatttgaaaatatggttgcattctgtgcatttgggcaaaatgagccaattcttctgtatatttacgatttccatgttgcccagcaggaggctgaaatgttcaaacgctacaatggaacatttccattacctggaatacaccaaagtcaagatgccctatgtacctgtccaaaactaccccatcaaggctcttttcagattgatgaatttgtccacactgaaagttcttcaacgaagatgcagctagtaaaacagaggcttgaaactgtcacagaggtgatacgttcctgtgctgcaaaagtcaacaaaaatctctcatttacttcaccaccagcagtttcctcacaacagtctaagttaaagcagtcaaacatgctgaccgctcaagagattctacatcagtttggtttcactcagaccgggattatcagcatagaaagaaagccttgtaaccatcaggtagatacagcaccaacggtagtggctctttatgactacacagcgaatcgatcagatgaactaaccatccatcgcggagacattatccgagtgtttttcaaagataatgaagactggtggtatggcagcataggaaagggacaggaaggttattttccagctaatcatgtggctagtgaaacactgtatcaagaactgcctcctgagataaaggagcgatcccctcctttaagccctgaggaaaaaactaaaatagaaaaatctccagctcctcaaaagcaatcaatcaataagaacaagtcccaggacttcagactaggctcagaatctatgacacattctgaaatgagaaaagaacagagccatgaggaccaaggacacataatggatacacggatgaggaagaacaagcaagcaggcagaaaagtcactctaatagagta-3′ FANCC (SEQ ID NO: 338)5′-atggctcaagattcagtagatctttcttgtgattatcagttttggatgcagaagctttctgtatgggatcaggcttccactttggaaacccagcaagacacctgtcttcacgtggctcagttccaggagttcctaaggaagatgtatgaagccttgaaagagatggattctaatacagtcattgaaagattccccacaattggtcaactgttggcaaaagcttgttggaatccttttattttagcatatgatgaaagccaaaaaattctaatatggtgcttatgttgtctaattaacaaagaaccacagaattctggacaatcaaaacttaactcctggatacagggtgtattatctcatatactttcagcactcagatttgataaagaagttgctcttttcactcaaggtcttgggtatgcacctatagattactatcctggtttgcttaaaaatatggttttatcattagcgtctgaactcagagagaatcatcttaatggatttaacactcaaaggcgaatggctcccgagcgagtggcgtccctgtcacgagtttgtgtcccacttattaccctgacagatgttgaccccctggtggaggctctcctcatctgtcatggacgtgaacctcaggaaatcctccagccagagttctttgaggctgtaaacgaggccattttgctgaagaagatttctctccccatgtcagctgtagtctgcctctggcttcggcaccttcccagccttgaaaaagcaatgctgcatctttttgaaaagctaatctccagtgagagaaattgtctgagaaggatcgaatgctttataaaagattcatcgctgcctcaagcagcctgccaccctgccatattccgggttgttgatgagatgttcaggtgtgcactcctggaaaccgatggggccctggaaatcatagccactattcaggtgtttacgcagtgctttgtagaagctctggagaaagcaagcaagcagctgcggtttgcactcaagacctactttccttacacttctccatctcttgccatggtgctgctgcaagaccctcaagatatccctcggggacactggctccagacactgaagcatatttctgaactgctcagagaagcagttgaagaccagactcatgggtcctgcggaggtccctttgagagctggttcctgttcattcacttcggaggatgggctgagatggtggcagagcaattactgatgtcggcagccgaaccccccacggccctgctgtggctcttggccttctactacggcccccgtgatgggaggcagcagagagcacagactatggtccaggtgaaggccgtgctgggccacctcctggcaatgtccagaagcagcagcctctcagcccaggacctgcagacggtagcaggacagggcacagacacagacctcagagctcctgcacaacagctgatcaggcaccttctcctcaacttcctgctctgggctcctggaggccacacgatcgcctgggatgtcatcaccctgatggctcacactgctgagataactcacgagatcattggctttcttgaccagaccttgtacagatggaatcgtcttggcattgaaagccctagatcagaaaaactggcccgagagctccttaaagagctgcgaactcaagtctag-3′ MYBPC3 (SEQ ID NO: 339)5′-atgcctgagccggggaagaagccagtctcagcttttagcaagaagccacggtcagtggaagtggccgcaggcagccctgccgtgttcgaggccgagacagagcgggcaggagtgaaggtgcgctggcagcgcggaggcagtgacatcagcgccagcaacaagtacggcctggccacagagggcacacggcatacgctgacagtgcgggaagtgggccctgccgaccagggatcttacgcagtcattgctggctcctccaaggtcaagttcgacctcaaggtcatagaggcagagaaggcagagcccatgctggcccctgcccctgcccctgctgaggccactggagcccctggagaagccccggccccagccgctgagctgggagaaagtgccccaagtcccaaagggtcaagctcagcagctctcaatggtcctacccctggagcccccgatgaccccattggcctcttcgtgatgcggccacaggatggcgaggtgaccgtgggtggcagcatcaccttctcagcccgcgtggccggcgccagcctcctgaagccgcctgtggtcaagtggttcaagggcaaatgggtggacctgagcagcaaggtgggccagcacctgcagctgcacgacagctacgaccgcgccagcaaggtctatctgttcgagctgcacatcaccgatgcccagcctgccttcactggcagctaccgctgtgaggtgtccaccaaggacaaatttgactgctccaacttcaatctcactgtccacgaggccatgggcaccggagacctggacctcctatcagccttccgccgcacgagcctggctggaggtggtcggcggatcagtgatagccatgaggacactgggattctggacttcagctcactgctgaaaaagagagacagtttccggaccccgagggactcgaagctggaggcaccagcagaggaggacgtgtgggagatcctacggcaggcacccccatctgagtacgagcgcatcgccttccagtacggcgtcactgacctgcgcggcatgctaaagaggctcaagggcatgaggcgcgatgagaagaagagcacagcctttcagaagaagctggagccggcctaccaggtgagcaaaggccacaagatccggctgaccgtggaactggctgaccatgacgctgaggtcaaatggctcaagaatggccaggagatccagatgagcggcagcaagtacatctttgagtccatcggtgccaagcgtaccctgaccatcagccagtgctcattggcggacgacgcagcctaccagtgcgtggtgggtggcgagaagtgtagcacggagctctttgtgaaagagccccctgtgctcatcacgcgccccttggaggaccagctggtgatggtggggcagcgggtggagtttgagtgtgaagtatcggaggagggggcgcaagtcaaatggctgaaggacggggtggagctgacccgggaggagaccttcaaataccggttcaagaaggacgggcagagacaccacctgatcatcaacgaggccatgctggaggacgcggggcactatgcactgtgcactagcgggggccaggcgctggctgagctcattgtgcaggaaaagaagctggaggtgtaccagagcatcgcagacctgatggtgggcgcaaaggaccaggcggtgttcaaatgtgaggtctcagatgagaatgttcggggtgtgtggctgaagaatgggaaggagctggtgcccgacagccgcataaaggtgtcccacatcgggcgggtccacaaactgaccattgacgacgtcacacctgccgacgaggctgactacagctttgtgcccgagggcttcgcctgcaacctgtcagccaagctccacttcatggaggtcaagattgacttcgtacccaggcaggaacctcccaagatccacctggactgcccaggccgcataccagacaccattgtggttgtagctggaaataagctacgtctggacgtccctatctctggggaccctgctcccactgtgatctggcagaaggctatcacgcaggggaataaggccccagccaggccagccccagatgccccagaggacacaggtgacagcgatgagtgggtgtttgacaagaagctgctgtgtgagaccgagggccgggtccgcgtggagaccaccaaggaccgcagcatcttcacggtcgagggggcagagaaggaagatgagggcgtctacacggtcacagtgaagaaccctgtgggcgaggaccaggtcaacctcacagtcaaggtcatcgacgtgccagacgcacctgcggcccccaagatcagcaacgtgggagaggactcctgcacagtacagtgggagccgcctgcctacgatggcgggcagcccatcctgggctacatcctggagcgcaagaagaagaagagctaccggtggatgcggctgaacttcgacctgattcaggagctgagtcatgaagcgcggcgcatgatcgagggcgtggtgtacgagatgcgcgtctacgcggtcaacgccatcggcatgtccaggcccagccctgcctcccagcccttcatgcctatcggtccccccagcgaacccacccacctggcagtagaggacgtctctgacaccacggtctccctcaagtggcggcccccagagcgcgtgggagcaggaggcctggatggctacagcgtggagtactgcccagagggctgctcagagtgggtggctgccctgcaggggctgacagagcacacatcgatactggtgaaggacctgcccacgggggcccggctgcttttccgagtgcgggcacacaatatggcagggcctggagcccctgttaccaccacggagccggtgacagtgcaggagatcctgcaacggccacggcttcagctgcccaggcacctgcgccagaccattcagaagaaggtcggggagcctgtgaaccttctcatccctttccagggcaagccccggcctcaggtgacctggaccaaagaggggcagcccctggcaggcgaggaggtgagcatccgcaacagccccacagacaccatcctgttcatccgggccgctcgccgcgtgcattcaggcacttaccaggtgacggtgcgcattgagaacatggaggacaaggccacgctggtgctgcaggttgttgacaagccaagtcctccccaggatctccgggtgactgacgcctggggtcttaatgtggctctggagtggaagccaccccaggatgtcggcaacacggagctctgggggtacacagtgcagaaagccgacaagaagaccatggagtggttcaccgtcttggagcattaccgccgcacccactgcgtggtgccagagctcatcattggcaatggctactacttccgcgtcttcagccagaatatggttggctttagtgacagagcggccaccaccaaggagcccgtctttatccccagaccaggcatcacctatgagccacccaactataaggccctggacttctccgaggccccaagcttcacccagcccctggtgaaccgctcggtcatcgcgggctacactgctatgctctgctgtgctgtccggggtagccccaagcccaagatttcctggttcaagaatggcctggacctgggagaagacgcccgcttccgcatgttcagcaagcagggagtgttgactctggagattagaaagccctgcccctttgacgggggcatctatgtctgcagggccaccaacttacagggcgaggcacggtgtgagtgccgcctggaggtgcgagtgcctcagtga-3′ IL2RG(SEQ ID NO: 340)5′-atgttgaagccatcattaccattcacatccctcttattcctgcagctgcccctgctgggagtggggctgaacacgacaattctgacgcccaatgggaatgaagacaccacagctgatttcttcctgaccactatgcccactgactccctcagtgtttccactctgcccctcccagaggttcagtgttttgtgttcaatgtcgagtacatgaattgcacttggaacagcagctctgagccccagcctaccaacctcactctgcattattggtacaagaactcggataatgataaagtccagaagtgcagccactatctattctctgaagaaatcacttctggctgtcagttgcaaaaaaaggagatccacctctaccaaacatttgttgttcagctccaggacccacgggaacccaggagacaggccacacagatgctaaaactgcagaatctggtgatcccctgggctccagagaacctaacacttcacaaactgagtgaatcccagctagaactgaactggaacaacagattcttgaaccactgtttggagcacttggtgcagtaccggactgactgggaccacagctggactgaacaatcagtggattatagacataagttctccttgcctagtgtggatgggcagaaacgctacacgtttcgtgttcggagccgctttaacccactctgtggaagtgctcagcattggagtgaatggagccacccaatccactgggggagcaatacttcaaaagagaatcctttcctgtttgcattggaagccgtggttatactgttggctccatgggattgattatcagccttctctgtgtgtatttctggctggaacggacgatgccccgaattcccaccctgaagaacctagaggatcttgttactgaataccacgggaacttttcggcctggagtggtgtgtctaagggactggctgagagtctgcagccagactacagtgaacgactctgcctcgtcagtgagattcccccaaaaggaggggcccttggggaggggcctggggcctccccatgcaaccagcatagcccctactgggcccccccatgttacaccctaaagcctgaaacctga-3′

DISCUSSION

Genome editing technologies are revolutionizing biomedical research.Highly active nucleases, such as zinc finger nucleases (ZFNs)¹,transcription activator-like effector nucleases (TALENs)²⁻⁴, and Casproteins of CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats) system⁵⁻⁷ have been successfully engineered to manipulate thegenome in a myriad of organisms. Recently, deaminases have beenharnessed to precisely change the genetic code without breakingdouble-stranded DNA. By coupling a cytidine or an adenosine deaminasewith the CRISPR-Cas9 system, researchers created programmable baseeditors that enable the conversion of C⋅G to T⋅A or A⋅T to G⋅C ingenomic DNA⁸⁻¹⁰, offering novel opportunities for correctingdisease-causing mutations.

Aside from DNA, RNA is an attractive target for genetic correctionbecause RNA modification could alter the protein function withoutgenerating any permanent changes to the genome. The ADAR adenosinedeaminases are currently exploited to achieve precise base editing onRNAs. Three kinds of ADAR proteins have been identified in mammals,ADAR1 (isoforms p110 and p150), ADAR2 and ADAR3 (catalyticinactive)^(11, 12), whose substrates are double-stranded RNAs, in whichan adenosine (A) mismatched with a cytosine (C) is preferentiallydeaminated to inosine (I). Inosine is believed to mimic guanosine (G)during translation^(13, 14). To achieve targeted RNA editing, the ADARprotein or its catalytic domain was fused with a λN peptide¹⁵⁻¹², aSNAP-tag¹⁸⁻²² or a Cas protein (dCas13b)²³, and a guide RNA was designedto recruit the chimeric ADAR protein to the specific site.Alternatively, overexpressing ADAR1 or ADAR2 proteins together with anR/G motif-bearing guide RNA was also reported to enable targeted RNAediting²⁴⁻²⁷.

All these reported nucleic acid editing methods in mammalian system relyon ectopic expression of two components: an enzyme and a guide RNA.Although these binary systems work efficiently in most studies, someinherent obstacles limit their broad applications, especially intherapies. Because the most effective in vivo delivery for gene therapyis through viral vectors²⁸, and the highly desirable adeno-associatedvirus (AAV) vectors are limited with cargo size (˜4.5 kb), making itchallenging for accommodating both the protein and the guideRNA^(29, 39). Over-expression of ADAR1 has recently been reported toconfer oncogenicity in multiple myelomas due to aberrant hyper-editingon RNAs³¹, and to generate substantial global off-targeting edits³². Inaddition, ectopic expression of proteins or their domains of non-humanorigin has potential risk of eliciting immunogenicity^(39, 33).Moreover, pre-existing adaptive immunity and p53-mediated DNA damageresponse may compromise the efficacy of the therapeutic protein, such asCas9³⁴⁻³⁸. Although it has been attempted to utilize endogenousmechanism for RNA editing, this was tried only by injectingpre-assembled target transcript:RNA duplex into Xenopus embryos³⁹.Alternative technologies for robust nucleic acid editing that don't relyon ectopic expression of proteins are much needed. Here, we developed anovel approach that leverages endogenous ADAR for RNA editing. We showedthat expressing a deliberately designed guide RNA enables efficient andprecise editing on endogenous RNAs, and corrects pathogenic mutations.This unary nucleic acid editing platform may open new avenues fortherapeutics and research.

In particular, we showed that expression of a linear arRNA with adequatelength is capable of guiding endogenous ADAR proteins to edit adenosineto inosine on the targeted transcripts. This system, referred to asLEAPER, utilizes endogenous ADAR proteins to achieve programmablenucleic acid editing, thus possessing advantages over existingapproaches.

The rare quality of LEAPER is its simplicity because it only relies on asmall size of RNA molecule to direct the endogenous proteins for RNAediting. This is reminiscent of RNAi, in which a small dsRNA couldinvoke native mechanism for targeted RNA degradation⁵¹. Because of thesmall size, arRNA could be readily delivered by a variety of viral andnon-viral vehicles. Different from RNAi, LEAPER catalyzes the precise Ato I switch without generating cutting or degradation of targetedtranscripts (FIG. 18A). Although the length requirement for arRNA islonger than RNAi, it neither induces immune-stimulatory effects at thecellular level (FIG. 22E, f and FIG. 29E) nor affects the function ofendogenous ADAR proteins (FIG. 22A, b), making it a safe strategy forRNA targeting. Remarkably, it has been reported that ectopic expressionof ADAR proteins or their catalytic domains induces substantial globaloff-target edits³² and possibly triggers cancer³¹.

Recently, several groups reported that cytosine base editor couldgenerate substantial off-target single-nucleotide variants in mouseembryos, rice or human cell lines due to the expression of an effectorprotein, which illustrates the advantage of LEAPER for potentialtherapeutic application⁵²⁻⁵⁴. Gratifyingly, LEAPER empowers efficientediting while elicits rare global off-target editing (FIG. 20 and FIG.21). In addition, LEAPER could minimize potential immunogenicity orsurmount delivery obstacles commonly shared by other methods thatrequire the introduction of foreign proteins.

For LEAPER, we would recommend using arRNA with a minimal size above70-nt to achieve desirable activity. In the native context, ADARproteins non-specifically edit Alu repeats which have a duplex of morethan 300-nt⁵⁵. Of note, Alu repeats form stable intramolecular duplex,while the LEAPER results in an intermolecular duplex between arRNA andmRNA or pre-mRNA, which is supposed to be less stable and more difficultto form. Therefore, we hypothesized that an RNA duplex longer than 70-ntis stoichiometrically important for recruiting or docking ADAR proteinsfor effective editing. Indeed, longer arRNA resulted in higher editingyield in both ectopically expressed reporters and endogenous transcripts(FIG. 16D and FIG. 17B). However, because ADAR proteins promiscuouslydeaminate adenosine base in the RNA duplex, longer arRNA may incur moreoff-targets within the targeting window.

While LEAPER could effectively target native transcripts, their editingefficiencies and off-target rates varied. For PPIB transcript-targeting,we could convert 50% of targeted adenosine to inosine without evidentoff-targets within the covering windows (FIG. 17B, f). The off-targetsbecame more severe for other transcripts. We have managed to reduceoff-targets such as introducing A-G mismatches or consecutive mismatchesto repress undesired editing. However, too many mismatches coulddecrease on-target efficiency. Weighing up the efficiency and potentialoff-targets, we would recommend arRNA with the length ranging from 100-to 150-nt for editing on endogenous transcripts. If there is a choice,it's better to select regions with less adenosine to minimize the chanceof unwanted edits. Encouragingly, we have not detected any off-targetsoutside of the arRNA-targeted-transcript duplexes (FIG. 20).

We have optimized the design of the arRNA to achieve improved editingefficiency and demonstrated that LEAPER could be harnessed to manipulategene function or correct pathogenic mutation. We have also shown thatLEAPER is not limited to only work on UAC instead that it works withpossibly any adenosine regardless of its flanking nucleotides (FIG. 16F,g and FIG. 17C). Such flexibility is advantageous for potentialtherapeutic correction of genetic diseases caused by certain singlepoint mutations. Interestingly, in editing the IDUA transcripts, thearRNA targeting pre-mRNA is more effective than that targeting matureRNA, indicating that nuclei are the main sites of action for ADARproteins and LEAPER could be leveraged to manipulate splicing bymodifying splice sites within pre-mRNAs. What's more, LEAPER hasdemonstrated high efficiency for simultaneously targeting multiple genetranscripts (FIG. 17D). This multiplexing capability of LEAPER might bedeveloped to cure certain polygenetic diseases in the future.

It is beneficial to perform genetic correction at the RNA level. First,editing on targeted transcripts would not permanently change the genomeor transcriptome repertoire, making RNA editing approaches safer fortherapeutics than means of genome editing. In addition, transientediting is well suited for temporal control of treating diseases causedby occasional changes in a specific state. Second, LEAPER and other RNAediting methods would not introduce DSB on the genome, avoiding the riskof generating undesirable deletions of large DNA fragments³⁷. DNA baseediting methods adopting nickase Cas9 could still generate indels in thegenome⁸. Furthermore, independent of native DNA repair machinery, LEAPERshould also work in post-mitosis cells such as cerebellum cells withhigh expression of ADAR2¹¹.

We have demonstrated that LEAPER could apply to a broad spectrum of celltypes such as human cell lines (FIG. 14C), mouse cell lines (FIG. 14D)and human primary cells including primary T cells (FIG. 27 and FIG.28D). Efficient editing through lentiviral delivery or synthesized oligoprovides increased potential for therapeutic development (FIG. 28).Moreover, LEAPER could produce phenotypic or physiological changes invarieties of applications including recovering the transcriptionalregulatory activity of p53 (FIG. 7), correcting pathogenic mutations(FIG. 26), and restoring the α-L-iduronidase activity in Hurler syndromepatient-derived primary fibroblasts (FIG. 29). It can thus be envisagedthat LEAPER has enormous potential in disease treatment.

Stafforst and colleagues reported a new and seemingly similar RNAediting method, named RESTORE, which works through recruiting endogenousADARs using synthetic antisense oligonucleotides⁵⁶. The fundamentaldifference between RESTORE and LEAPER lies in the distinct nature of theguide RNA for recruiting endogenous ADAR. The guide RNA of RESTORE islimited to chemosynthetic antisense oligonucleotides (ASO) depending oncomplex chemical modification, while arRNA of LEAPER can be generated ina variety of ways, chemical synthesis and expression from viral ornon-viral vectors (FIG. 28 and FIG. 29). Importantly, being heavilychemically modified, ASOs is restricted to act transiently in diseasetreatment. In contrast, arRNA could be produced through expression, afeature particularly important for the purpose of constant editing.

There are still rooms for improvements regarding LEAPER's efficiency andspecificity. Because LEAPER relies on the endogenous ADAR, theexpression level of ADAR proteins in target cells is one of thedeterminants for successful editing. According to previous report⁵⁷ andour observations (FIG. 14A, b), the ADAR1^(p110) is ubiquitouslyexpressed across tissues, assuring the broad applicability of LEAPER.The ADAR1^(p150) is an interferon-inducible isoform⁵⁸, and has proven tobe functional in LEAPER (FIG. 11E, FIG. 12B). Thus, co-transfection ofinterferon stimulatory RNAs with the arRNA might further improve editingefficiency under certain circumstances. Alternatively, as ADAR3 playsinhibitory roles, inhibition of ADAR3 might enhance editing efficiencyin ADAR3-expressing cells. Moreover, additional modification of arRNAmight increase its editing efficiency. For instance, arRNA fused withcertain ADAR-recruiting scaffold may increase local ADAR proteinconcentration and consequently boost editing yield. So far, we couldonly leverage endogenous ADAR1/2 proteins for the A to I baseconversion. It is exciting to explore whether more native mechanismscould be harnessed similarly for the modification of genetic elements,especially to realize potent nucleic acid editing.

Altogether, we provided a proof of principle that the endogenousmachinery in cells could be co-opted to edit RNA transcripts. Wedemonstrated that LEAPER is a simple, efficient and safe system,shedding light on a novel path for gene editing-based therapeutics andresearch.

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1. A deaminase-recruiting RNA (dRNA) of 60-200 nucleotides, wherein: a)the dRNA comprises a complementary RNA sequence capable of hybridizingto a target RNA; b) the dRNA is capable of recruiting a deaminase or aconstruct comprising a deaminase or a construct comprising a catalyticdomain of a deaminase to deaminate a target adenosine in the target RNA;and c) the dRNA comprises one or more chemical modifications.
 2. ThedRNA of claim 1, wherein the dRNA is longer than about any of 60 nt, 65nt, 70 nt, 80 nt, 90 nt, 100 nt, or 110 nt.
 3. The dRNA of claim 1,comprising one or more mismatches, wobbles and/or bulges with thecomplementary target RNA region.
 4. The dRNA of claim 1, wherein thecomplementary RNA sequence comprises a cytidine, adenosine or uridinedirectly opposite to a target adenosine in the target RNA.
 5. The dRNAof claim 4, wherein the cytidine, adenosine or uridine directly oppositeto the target adenosine locates at least about 7 nucleotides away fromthe 3′ end.
 6. The dRNA of claim 4, wherein the cytidine, adenosine oruridine directly opposite to the target adenosine locates at least about25 nucleotides away from the 5′ end.
 7. The dRNA of claim 4, wherein thelengths of the 5′ and 3′ sequences flanking the cytidine, adenosine oruridine directly opposite to the target adenosine are unequal.
 8. ThedRNA of claim 4, wherein the length of the 5′ sequence flanking thecytidine, adenosine or uridine directly opposite to the target adenosineis longer than the 3′ sequence.
 9. The dRNA of claim 1, comprising acytidine directly opposite to the target adenosine in the target RNA.10. The dRNA of claim 1, wherein the complementary RNA sequencecomprises one or more guanosines each opposite to a non-target adenosinein the target RNA.
 11. The dRNA of claim 1, wherein the complementarysequence comprises two or more consecutive mismatch nucleotides oppositeto a non-target adenosine in the target RNA. 12-13. (canceled)
 14. ThedRNA of claim 1, wherein the three-base motif is UAG, and wherein thedRNA comprises an A directly opposite to the uridine in the three-basemotif, a cytidine directly opposite to the target adenosine, and acytidine, guanosine or uridine directly opposite the guanosine in thethree-base motif.
 15. The dRNA of claim 14, comprising a 5′-CCA-3′directly opposite to the three-base motif of UAG.
 16. The dRNA of claim1, wherein the chemical modification comprises methylation and/orphosphorothioation.
 17. The dRNA of claim 16, wherein the chemicalmodification comprises 2′-O-methylation and/or internucleotidephosphorothioate linkage.
 18. The dRNA of claim 16, wherein the chemicalmodification comprises a 2′-O-methylationin the first and last 1-5, 2-5,3-5, 4-5 nucleotides and/or phosphorothioations in the first and last1-5, 2-5, 3-5, 4-5 internucleotide linkages.
 19. The dRNA of claim 16,wherein the chemical modification comprises a 2′-O-methylation and/or a3′-phosphorothioation in the nucleotide opposite to the target adenosineand/or its 5′ and/or 3′ most adjacent nucleotides.
 20. The dRNA of claim1, wherein the chemical modification is selected from a group consistingof: 1) 2′-O-methylations in the first and last 3 nucleotides and/orphosphorothiations in the first and last 3 internucleotide linkages; 2)2′-O-methylations in the first and last 3 nucleotides and/orphosphorothiations in the first and last 3 internucleotide linkages, and2′-O-methylations in a single or multiple or all uridines; 3)2′-O-methylations in the first and last 3 nucleotides,phosphorothiations in the first and last 3 internucleotide linkages,2′-O-methylations in a single or multiple or all uridines, and amodification in the nucleotide opposite to the target adenosine, and/orits 5′ and/or 3′ most adjacent nucleotides; 4) 2′-O-methylations in thefirst and last 3 nucleotides, phosphorothiations in the first and last 3internucleotide linkages, 2′-O-methylations in a single or multiple orall uridines, and 2′-O-methylation in the nucleotide most adjacent tothe 3′ terminus and/or 5′ terminus of the nucleotide opposite to thetarget adenosine; 5) 2′-O-methylations in the first and last 3nucleotides, phosphorothiations in the first and last 3 internucleotidelinkages, 2′-O-methylations in a single or multiple or all uridines, andphosphorothiation linkage in the nucleotide opposite to the targetadenosine and/or its 5′ and/or 3′ most adjacent nucleotides; and 6)2′-O-methylations in the first and last 1-5 nucleotides and/orphosphorothiations in the first and last 1-5 internucleotide linkages.21. The dRNA of claim 20, wherein the modification in the nucleotideopposite to the target adenosine, and/or one or two nucleotides mostadjacent to the nucleotide opposite to the target adenosine is2′-O-methylation and/or phosphorothiation linkage.
 22. The dRNA of claim1, which does not comprise an ADAR-recruiting domain capable of formingan intramolecular stem loop structure for binding an ADAR enzyme.
 23. Aconstruct comprising or encoding a dRNA of claim
 1. 24. A method forediting a target RNA in a host cell, comprising introducing a dRNA ofclaim 1 into host cells.
 25. The method of claim 24, further comprisesintroducing an inhibitor of ADAR3 to the host cell.
 26. The method ofclaim 24, further comprises introducing a stimulator of interferon tothe host cell.
 27. The method of claim 24, comprising introducing aplurality of the dRNAs each targeting a different target RNA. 28.(canceled)
 29. The method of claim 24, further comprises introducing anexogenous ADAR to the host cell.
 30. The method of claim 29, wherein theADAR is an ADAR1 comprising an E1008 mutation.
 31. A construction,composition, cell, library or kit comprising the dRNAs of claim 1.